Ribosome structure and protein synthesis inhibitors

ABSTRACT

The invention provides methods for producing high resolution crystals of ribosomes and ribosomal subunits as well as crystals produced by such methods. The invention also provides high resolution structures of ribosomal subunits either alone or in combination with protein synthesis inhibitors. The invention provides methods for identifying ribosome-related ligands and methods for designing ligands with specific ribosome-binding properties as well as ligands that may act as protein synthesis inhibitors. Thus, the methods and compositions of the invention may be used to produce ligands that are designed to specifically kill or inhibit the growth of any target organism.

RELATED APPLICATIONS

[0001] This application is a continuation-in-part of U.S. applicationSer. No. 09/635,708, filed Aug. 9, 2000, and claims the benefit of (i)U.S. Provisional Application No. 60/223,977, filed Aug. 9, 2000, (ii)U.S. Provisional Application No. [Atty. Docket No. RIB-002PR], entitled“The Kink-Turn: a New RNA Secondary Structure Motif,” filed Jul. 20,2001, and (iii) U.S. Provisional Application No. [Atty. Docket No.RIB-002PR2], entitled “The Kink-Turn: a New RNA,” filed Aug. 1, 2001,the disclosures of each of the foregoing of which are incorporated byreference herein.

GOVERNMENT LICENSE RIGHTS

[0002] Certain work described herein was supported, in part, by FederalGrant Nos. NIH-GM22778 and NIH-GM54216, awarded by the NationalInstitutes of Health. The Government may have certain rights in theinvention.

FIELD OF THE INVENTION

[0003] The present invention relates generally to the field of proteinbiosynthesis and to modulators, for example, inhibitors, of proteinbiosynthesis. More particularly, the invention relates to methods andcompositions for elucidating the three-dimensional structure of thelarge ribosomal subunit, either alone or in combination with a proteinsynthesis inhibitor; the three-dimensional structure of the largeribosomal subunit, either alone or in combination with a proteinsynthesis inhibitor; the use of such structures in the design andtesting of novel protein synthesis inhibitors; and novel proteinsynthesis inhibitors.

BACKGROUND I. Ribosomes: Structure, Function, and Composition

[0004] Ribosomes are ribonucleoproteins which are present in bothprokaryotes and eukaryotes. They comprise about two-thirds RNA andone-third protein. Ribosomes are the cellular organelles responsible forprotein synthesis. During gene expression, ribosomes translate thegenetic information encoded in a messenger RNA into protein (Garrett etal. (2000) “The Ribosome: Structure, Function, Antibiotics and CellularInteractions,” American Society for Microbiology, Washington, D.C.).

[0005] Ribosomes comprise two nonequivalent ribonucleoprotein subunits.The larger subunit (also known as the “large ribosomal subunit”) isabout twice the size of the smaller subunit (also known as the “smallribosomal subunit”). The small ribosomal subunit binds messenger RNA(mRNA) and mediates the interactions between mRNA and transfer RNA(tRNA) anticodons on which the fidelity of translation depends. Thelarge ribosomal subunit catalyzes peptide bond formation—thepeptidyl-transferase reaction of protein synthesis—and includes (atleast) two different tRNA binding sites: the A-site which accommodatesthe incoming aminoacyl-tRNA, which is to contribute its amino acid tothe growing peptide chain, and the P-site which accommodates thepeptidyl-tRNA complex, i.e., the tRNA linked to all the amino acids thathave so far been added to the peptide chain. The large ribosomal subunitalso includes one or more binding sites for G-protein factors thatassist in the initiation, elongation, and termination phases of proteinsynthesis. The large and small ribosomal subunits behave independentlyduring the initiation phase of protein synthesis; however, they assembleinto complete ribosomes when elongation is about to begin.

[0006] The molecular weight of the prokaryotic ribosome is about 2.6×10⁶daltons. In prokaryotes, the small ribosomal subunit contains a 16S(Svedberg units) ribosomal RNA (rRNA) having a molecular weight of about5.0×10⁵ daltons. The large ribosomal subunit contains a 23S rRNA havinga molecular weight of about 1.0×10⁶ daltons and a 5S rRNA having amolecular weight of about 4.0×10⁵ daltons. The prokaryotic small subunitcontains about 20 different proteins and its large subunit containsabout 35 proteins. The large and small ribosomal subunits togetherconstitute a 70S ribosome in prokaryotes.

[0007] Eukaryotic ribosomes generally are bigger than their prokaryoticcounterparts. In eukaryotes, the large and small subunits together makean 80S ribosome. The small subunit of a eukaryotic ribosome includes asingle 18S rRNA, while the large subunit includes a 5S rRNA, a 5.8SrRNA, and a 28S rRNA. The 5.8S rRNA is structurally related to the 5′end of the prokaryotic 23S rRNA, and the 28S rRNA is structurallyrelated to the remainder of the prokaryotic 23S rRNA (Moore (1998) Annu.Rev. Biophys. 27: 35-58). Eukaryotic ribosomal proteins arequalitatively similar to the prokaryotic ribosomal proteins; however,the eukaryotic proteins are bigger and more numerous (Moore (1998)supra).

II. Structural Conservation of the Large Ribosomal Subunit

[0008] While the chemical composition of large ribosomal subunits varyfrom species to species, the sequences of their components provideunambiguous evidence that they are similar in three-dimensionalstructure, function in a similar manner, and are related evolutionarily.The evolutionary implications of rRNA sequence data available arereviewed in the articles of Woese and others in part II of RibosomalRNA, Structure, Evolution, Processing and Function in ProteinBiosynthesis, (Zimmermann and Dahlberg, eds., CRC Press, Boca Raton,Fla., 1996). The article by Garret and Rodriguez-Fonseca in part IV ofthe same volume discusses the unusually high level of sequenceconservation observed in the peptidyl transferase region of the largeribosomal subunit. The ribosomes of archeal species like Haloarculamarismortui resemble those obtained from eubacterial species like E.coli in size and complexity. However, the proteins in H. marismortuiribosomes are more closely related to the ribosomal proteins found ineukaryotes (Wool et al. (1995) Biochem. Cell Biol. 73: 933-947).

III. Determination of the Structure of Ribosomes

[0009] Much of what is known about ribosome structure is derived fromphysical and chemical methods that produce relatively low-resolutioninformation. Electron microscopy (EM) has contributed to anunderstanding of ribosome structure ever since the ribosome wasdiscovered. In the 1970s, low resolution EM revealed the shape andquaternary organization of the ribosome. By the end of 1980s, thepositions of the surface epitopes of all the proteins in the E. colismall subunit, as well as many in the large subunit, had been mappedusing immunoelectron microscopy techniques (Oakes et al. (1986),Structure, Function and Genetics of Ribosomes, (Hardesty, B. and Kramer,G., eds.) Springer-Verlag, New York, N.Y., pp. 47-67; Stoeffler et al.(1986), Structure, Function and Genetics of Ribosomes, (Hardesty, B. andKramer, G., eds.) Springer-Verlag, New York, N.Y., pp.28-46). In thelast few years, advances in single-particle cryo-EM and imagereconstruction have led to three-dimensional reconstructions of the E.coli 70S ribosome and its complexes with tRNAs and elongation factors toresolutions of between 15 Å and 25 Å (Stark et al. (1995) Structure 3:815-821; Stark et al. (1997) Nature 3898: 403-406; Agrawal et al. (1996)Science 271: 1000-1002; Stark et al. (1997) Cell 28: 19-28).Additionally, three-dimensional EM images of the ribosome have beenproduced at resolutions sufficiently high so that many of the proteinsand nucleic acids that assist in protein synthesis can be visualizedbound to the ribosome. An approximate model of the RNA structure in thelarge subunit has been constructed to fit a 7.5 Å resolution electronmicroscopic map of the 50S subunit from E. coli and availablebiochemical data (Mueller et al. (2000) J. Mol. Biol. 298: 35-59).

[0010] While the insights provided by EM have been useful, it has longbeen recognized that a full understanding of ribosome structure wouldderive only from X-ray crystallography. In 1979, Yonath and Wittmanobtained the first potentially useful crystals of ribosomes andribosomal subunits (Yonath et al. (1980) Biochem. Internat. 1: 428-435).By the mid 1980s, scientists were preparing ribosome crystals for X-raycrystallography (Maskowski et al. (1987) J. Mol. Biol. 193: 818-822).The first crystals of 50S ribosomal subunit from H. marismortui wereobtained in 1987. In 1991, improvements were reported in the resolutionof the diffraction data obtainable from the crystals of the 50Sribosomal subunit of H. marismortui (van Bohlen, K. (1991) J. Mol. Biol.222: 11).

[0011] In 1995, low resolution electron density maps for the large andsmall ribosomal subunits from halophilic and thermophilic sources werereported (Schlunzen et al. (1995) Biochem. Cell Biol. 73: 739-749).However, these low resolution electron density maps proved to bespurious (Ban et al. (1998) Cell 93: 1105-1115).

[0012] The first electron density map of the ribosome that showedfeatures recognizable as duplex RNA was a 9 Å resolution X-raycrystallographic map of the large subunit from Haloarcula marismortui(Ban et al. (1998) supra). Extension of the phasing of that map to 5 Åresolution made it possible to locate several proteins and nucleic acidsequences, the structures of which had been determined independently(Ban et al. (1999) Nature 400: 841-847).

[0013] At about the same time, using similar crystallographicstrategies, a 7.8 Å resolution map was generated of the entire Thermusthermophilus ribosome showing the positions of tRNA molecules bound toits A-, P-, and E- (protein exit site) sites (Cate et al. (1999) Science285: 2095-2104), and a 5.5 Å resolution map of the 30S subunit from T.thermophilus was obtained that allowed the fitting of solved proteinstructures and the interpretation of some of its RNA features (Clemons,Jr. et al. (1999) Nature 400: 833-840). Subsequently, a 4.5 Å resolutionmap of the T. thermophilus 30S subunit was published, which was based inpart on phases calculated from a model corresponding to 28% of thesubunit mass that had been obtained using a 6 Å resolution experimentalmap (Tocilj et al. (1999) Proc. Natl. Acad. Sci. USA 96: 14252-14257).

IV. Location of the Peptidyl Transferase Site in the Large RibosomalSubunit

[0014] It has been known for about 35 years that the peptidyltransferase activity responsible for the peptide bond formation thatoccurs during messenger RNA-directed protein synthesis is intrinsic tothe large ribosomal subunit (Traut et al. (1964) J. Mol. Biol. 10: 63;Rychlik (1966) Biochim. Biophys. Acta 114: 425; Monro (1967) J. Mol.Biol. 26: 147-151; Maden et al. (1968) J. Mol. Biol. 35: 333-345) and ithas been understood for even longer that the ribosome contains proteinsas well as RNA. In certain species of bacteria, for example, the largeribosomal subunit contains about 35 different proteins and two RNAs(Noller (1984) Ann. Rev. Biochem. 53: 119-162; Wittmann-Liebold et al.(1990) The Ribosome: Structure, Function, and Evolution, (W. E. Hill etal., eds.) American Society for Microbiology, Washington, D.C. (1990),pp. 598-616). These findings posed three related questions. Which of thealmost 40 macromolecular components of the large ribosomal subunitcontribute to its peptidyl transferase site, where is that site locatedin the large subunit, and how does it work?

[0015] By 1980, the list of components that might be part of theribosome's peptidyl transferase had been reduced to about half a dozenproteins and 23S rRNA (see Cooperman (1980) Ribosomes: Structure,Function and Genetics, (G. Chambliss et al., eds.) University ParkPress, Baltimore, Md. (1980), 531-554), and following the discovery ofcatalytic RNAs (Guerrier-Takada et al. (1983) Cell 35: 849-857; Krugeret al. (1982) Cell 31: 147-157), the hypothesis that 23S rRNA might beits sole constituent, which had been proposed years earlier, began togain favor. In 1984, Noller and colleagues published affinity labelingresults which showed that U2619 and U2620 (in E. coli: U2584, U2585) areadjacent to the CCA-end of P-site-bound tRNA (Barta et al. (1984) Proc.Nat. Acad. Sci. USA 81: 3607-3611; Vester et al. (1988) EMBO J. 7:3577-3587). These nucleotides appear to be part of a highly conservedinternal loop in the center of domain V of 23S rRNA. The hypothesis thatthis loop is intimately involved in the peptidyl transferase activitywas supported by the observation that mutations in that loop rendercells resistant to many inhibitors of peptidyl transferase, and evidenceimplicating it in this activity has continued to mount (see, Noller(1991) Ann. Rev. Biochem. 60: 191-227; Garrett et al. (1996) RibosomalRNA: Structure, Evolution, Processing and Function in ProteinBiosynthesis, (R. A. Zimmerman and A. E. Dahlberg, eds.) CRC Press, BocaRaton, Fla. (1996), pp. 327-355).

[0016] Definitive proof that the central loop in domain V is the solecomponent of the ribosome involved in the peptidyl transferase activityhas remained elusive, however. Studies have shown that it was possibleto prepare particles that retained peptidyl transferase activity byincreasingly vigorous deproteinizations of large ribosomal subunits,however, it was not possible to produce active particles that werecompletely protein-free. Nevertheless, combined with earlierreconstitution results (Franceschi et al. (1990) J. Biol. Chem. 265:6676-6682), this work reduced the number of proteins that might beinvolved to just two: L2 and L3 (see, Green et al. (1997) Annu. Rev.Biochem. 66: 679-716). More recently, Watanabe and coworkers reportedsuccess in eliciting peptidyl transferase activity from in vitrosynthesized, protein-free 23S rRNA (Nitta et al. (1998) RNA 4: 257-267),however, their observations appear not to have withstood furtherscrutiny. Thus the question still remained: is the ribosome a ribozymeor is it not?

[0017] Over the years, the location of the peptidyl transferase site inthe ribosome has been approached almost exclusively by electronmicroscopy. In the mid-1980s evidence that there is a tunnel runningthrough the large ribosomal subunit from the middle of its subunitinterface side to its back (Milligan et al. (1986) Nature 319: 693-695;Yonath et al. (1987) Science 236: 813-816) began to accumulate, andthere has been strong reason to believe that polypeptides pass throughit as they are synthesized (Bernabeu et al. (1982) Proc. Nat. Acad. Sci.USA 79: 3111-3115; Ryabova et al. (1988) FEBS Letters 226: 255-260;Beckmann et al. (1997) Science 278: 2123-2126). More recent cryo-EMinvestigations (Frank et al. (1995) Nature 376: 441-444; Frank et al.(1995) Biochem. Cell Biol. 73: 757-765; Stark et al. (1995) supra)confirmed the existence of the tunnel and demonstrated that the CCA-endsof ribosome-bound tRNAs bound to the A- and P-sites are found in thesubunit interface end of the tunnel. Consequently, the peptidyltransferase site must be located at that same position, which is at thebottom of a deep cleft in the center of the subunit interface surface ofthe large subunit, immediately below its central protuberance.

[0018] The substrates of the reaction catalyzed at the peptidyltransferase site of the large subunit are an aminoacyl-tRNA (aa-tRNA)and a peptidyl-tRNA. The former binds in the ribosome's A-site and thelatter in its P-site. The α-amino group of the aa-tRNA attacks thecarbon of the carbonyl acylating the 3′ hydroxyl group of thepeptidyl-tRNA, and a tetrahedral intermediate is formed at the carbonylcarbon. The tetrahedral intermediate resolves to yield a peptideextended by one amino acid esterified to the A-site bound tRNA and adeacylated tRNA in the P-site.

[0019] This reaction scheme is supported by the observations of Yarusand colleagues who synthesized an analogue of the tetrahedralintermediate by joining an oligonucleotide having the sequence CCdA topuromycin via a phosphoramide group (Welch et al. (1995) Biochemistry34: 385-390). The sequence CCA, which is the 3′ terminal sequence of alltRNAs, binds to the large subunit by itself, consistent with thebiochemical data showing that the interactions between tRNAs and thelarge subunit largely depend on their CCA sequences (Moazed et al.(1991) Proc. Natl. Acad. Sci. USA 88: 3725-3728). Puromycin is an α-tRNAanalogue that interacts with the ribosomal A-site, and the phosphoramidegroup of the compound mimics the tetrahedral carbon intermediate. Thistransition state analogue, CCdA-phosphate-puromycin (CCdA-p-Puro), bindstightly to the ribosome, and inhibits its peptidyl transferase activity(Welch et al. (1995) supra).

V. Structure Determination of Macromolecules Using X-ray Crystallography

[0020] In order to better describe efforts undertaken to determine thestructure of ribosomes, a general overview of X-ray crystallography isprovided below.

[0021] Each atom in a crystal scatters X-rays in all directions, butcrystalline diffraction is observed only when a crystal is orientedrelative to the X-ray beam so that the atomic scattering interferesconstructively. The orientations that lead to diffraction may becomputed if the wavelength of the X-rays used and the symmetry anddimensions of the crystal's unit cell are known (Blundell et al. (1976)Protein Crystallography (Molecular Biology Series), Academic Press,London). The result is that if a detector is placed behind a crystalthat is being irradiated with monochromatic X-rays of an appropriatewavelength, the diffraction pattern recorded will consist of spots, eachspot representing one of the orientations that gives rise toconstructive interference.

[0022] Each spot in such a pattern, however it is recorded, ischaracterized by (i) an intensity (its blackness); (ii) a location,which encodes the information about diffraction orientation; and (iii) aphase. If all of those things are known about each spot in a crystaldiffraction pattern, the distribution of electrons in the unit cell ofthe crystal may be computed by Fourier transformation (Blundell et al.(1976) supra), and from that distribution or electron density map,atomic positions can be determined.

[0023] Unfortunately, the phase information essential for computingelectron distributions cannot be measured directly from diffractionpatterns. One of the methods routinely used to determine the phases ofmacromolecules, such as proteins and nucleic acids, is called multipleisomorphous replacement (MIR) which involves the introduction of newX-ray scatterers into the unit cell of the crystal. Typically, theseadditions are heavy atoms, which make a significant contribution to thediffraction pattern. It is important that the additions be sufficientlylow in number so that their positions can be located and that they leavethe structure of the molecule or of the crystal cell unaltered, i.e.,the crystals should be isomorphous. Isomorphous replacement usually isperformed by diffusing different heavy-metal complexes into the channelsof the preformed protein crystals. Macromolecules expose side chains(such as SH groups) in these solvent channels that are able to bindheavy metals. It is also possible to replace endogenous light metals inmetalloproteins with heavier ones, e.g. zinc by mercury, or calcium bysamarium. Alternatively, the isomorphous derivative can be obtained bycovalently attaching a heavy metal to the macromolecule in solution andthen subjecting it to crystallization conditions.

[0024] Heavy metal atoms routinely used for isomorphous replacementinclude but are not limited to mercury, uranium, platinum, gold, lead,and selenium. Specific examples include mercury chloride, ethyl-mercuryphosphate, and osmium pentamine, iridium pentamine. Since such heavymetals contain many more electrons than the light atoms (H, N, C, O, andS) of the protein, the heavy metals scatter x-rays more strongly. Alldiffracted beams would therefore increase in intensity after heavy-metalsubstitution if all interference were positive. In fact, however, someinterference is negative; consequently, following heavy-metalsubstitution, some spots increase in intensity, others decrease, andmany show no detectable difference.

[0025] Phase differences between diffracted spots can be determined fromintensity changes following heavy-metal substitution. First, theintensity differences are used to deduce the positions of the heavyatoms in the crystal unit cell. Fourier summations of these intensitydifferences give maps, of the vectors between the heavy atoms, theso-called Patterson maps. From these vector maps, the atomic arrangementof the heavy atoms is deduced. From the positions of the heavy metals inthe unit cell, the amplitudes and phases of their contribution to thediffracted beams of protein crystals containing heavy metals iscalculated.

[0026] This knowledge then is used to find the phase of the contributionfrom the protein in the absence of the heavy-metal atoms. As both thephase and amplitude of the heavy metals and the amplitude of the proteinalone is known, as well as the amplitude of the protein plus heavymetals (i.e., protein heavy-metal complex), one phase and threeamplitudes are known. From this, the interference of the X-raysscattered by the heavy metals and protein can be calculated to determineif the interference is constructive or destructive. The extent ofpositive or negative interference, with knowledge of the phase of theheavy metal, give an estimate of the phase of the protein. Because twodifferent phase angles are determined and are equally good solutions, asecond heavy-metal complex can be used which also gives two possiblephase angles. Only one of these will have the same value as one of thetwo previous phase angles; it therefore represents the correct phaseangle. In practice, more than two different heavy-metal complexes areusually made in order to give a reasonably good estimate of the phasefor all reflections. Each individual phase estimate containsexperimental errors arising from errors in the measured amplitudes.Furthermore, for many reflections, the intensity differences are toosmall to measure after one particular isomorphous replacement, andothers can be tried.

[0027] The amplitudes and the phases of the diffraction data from theprotein crystals are used to calculate an electron-density map of therepeating unit of the crystal. This map then is interpreted toaccommodate the residues of the molecule of interest. Thatinterpretation is made more complex by several limitations in the data.First, the map itself contains errors, mainly due to errors in the phaseangles. In addition, the quality of the map depends on the resolution ofthe diffraction data, which, in turn, depends on how well-ordered thecrystals are. This directly influences the quality of the map that canbe produced. The resolution is measured in angstrom units (Å); thesmaller this number is, the higher the resolution and, therefore, thegreater the amount of detail that can be seen.

[0028] Building the initial model is a trial-and-error process. First,one has to decide how a polypeptide chain or nucleic acid weaves its waythrough the electron-density map. The resulting chain trace constitutesa hypothesis by which one tries to match the density of side chains tothe known sequence of the polypeptide or nucleic acid. When a reasonablechain trace has finally been obtained, an initial model is built thatfits the atoms of the molecule into the electron density. Computergraphics are used both for chain tracing and for model building topresent the data and manipulated the models.

[0029] The initial model will contain some errors. Provided the crystalsdiffract to high enough resolution (e.g., better than 3.5 Å), most orsubstantially all of the errors can be removed by crystallographicrefinement of the model using computer algorithms. In this process, themodel is changed to minimize the difference between the experimentallyobserved diffraction amplitudes and those calculated for a hypotheticalcrystal containing the model (instead of the real molecule). Thisdifference is expressed as an R factor (residual disagreement) which is0.0 for exact agreement and about 0.59 for total disagreement.

[0030] In general, the R factor for a well-determined macromolecularstructure preferably lies between 0.15 and 0.35 (such as less than about0.24-0.28). The residual difference is a consequence of errors andimperfections in the data. These derive from various sources, includingslight variations in the conformation of the protein molecules, as wellas inaccurate corrections both for the presence of solvent and fordifferences in the orientation of the microcrystals from which thecrystal is built. This means that the final model represents an averageof molecules that are slightly different both in conformation andorientation.

[0031] In refined structures at high resolution, there are usually nomajor errors in the orientation of individual residues, and theestimated errors in atomic positions are usually around 0.1-0.2 Å,provided the sequence of the protein or nucleic acid is known. Hydrogenbonds, both within the molecule of interest and to bound ligands, can beidentified with a high degree of confidence.

[0032] Typically, X-ray structures can be determined provided theresolution is better than 3.5 Å. Electron-density maps are interpretedby fitting the known amino acid and/or nucleic acid sequences intoregions of electron density.

VI. The Need for Higher Resolution for the 50S Ribosomal Subunit

[0033] Although the art provides crystals of the 50S ribosomal subunit,and 9 Å and 5 Å resolution X-ray crystallographic maps of the structureof the 50S ribosome, the prior art crystals and X-ray diffraction dataare not sufficient to establish the three-dimensional structures of all31 proteins and 3,043 nucleotides of the 50S ribosomal subunit. Thus,the prior art crystals and maps are inadequate for the structure-baseddesign of active agents, such as herbicides, drugs, insecticides, andanimal poisons.

[0034] More detailed, higher resolution X-ray crystallographic maps arenecessary in order to determine the location and three-dimensionalstructure of the proteins and nucleotides in ribosomes and ribosomalsubunits, particularly for the 50S ribosomal subunit. An accuratemolecular structure of the 50S ribosomal subunit will not only enablefurther investigation and understanding of the mechanism of proteinsynthesis, but also the development of effective therapeutic agents anddrugs that modulate (e.g., induce or inhibit) protein synthesis.

SUMMARY OF THE INVENTION

[0035] The present invention is based, in part, upon the determinationof a high resolution atomic structure of a ribosomal subunit, moreparticularly, a large subunit of a ribosome. The high resolutionstructure has been determined for a large ribosomal subunit present inthe organism, Haloarcula marismortui. However, in view of the high levelof sequence and structural homology between ribosomes of organisms indifferent kingdoms, the structural information disclosed herein can beused to produce, using routine techniques, high resolution structuralmodels of large ribosomal units for any organism of interest.

[0036] Although there is significant homology between ribosomes ofdifferent organisms, for example, between ribosomes of humans andcertain human pathogens, there still are differences that can beexploited therapeutically. For example, many clinically and commerciallysignificant protein synthesis inhibitors, for example, antibiotics suchas streptomycin, tetracycline, chloramphenicol and erythromycin,selectively target bacterial ribosomes and disrupt bacterial proteinsynthesis but at the same time do not target or otherwise significantlyaffect human ribosome function. As a result, over the years antibioticshave proven to be invaluable in the treatment of microbial infections inhumans. However, there is still an ongoing need for new proteinsynthesis inhibitors, particularly because of the development of strainsof pathogens that are resistant to known antibiotics. The informationprovided herein provides insights into the design of new proteinsynthesis inhibitors.

[0037] The invention herein provides methods and compositions forresolving to a high resolution, the three-dimensional structure of aribosomal subunit of interest. In addition, the invention providescomputer systems containing atomic co-ordinates that define at least aportion of the three-dimensional structure of a ribosome, morespecifically, a large ribosomal subunit. In addition, the inventionprovides methods of using the atomic co-ordinates to identify newmolecules that can selectively bind ribosomes, and that preferably actas selective inhibitors of protein synthesis. In addition, the inventionprovides new families of protein synthesis inhibitors. Each of theseaspects of the invention is discussed in more detail below.

[0038] In one aspect, the invention provides crystals, preferablyuntwinned crystals, of ribosomes or ribosomal subunits that have anaverage thickness greater than about 15 μm. More specifically, theinvention provides crystals having an average thickness from about 16 μmto about 65 μm, or from about 66 μm to about 105 μm, or from about 104μm to about 155 μm, or from about 156 μm to about 205 μm. In particular,the invention provides crystals having an average thickness from about100 μm to about 200 μm.

[0039] In a preferred embodiment, the invention provides crystals thathave an average thickness greater than about 15 μm and/or are untwinnedwherein the crystals comprise the large ribosomal subunit. Moreparticularly, the present invention provides such crystals wherein thelarge ribosomal subunit is a 50S or 60S ribosomal subunit. The crystalsmay be obtained using the ribosomes or ribosomal subunits fromprokaryotes or from eukaryotes. In a preferred embodiment, the inventionprovides crystals containing ribosomes or ribosomal subunits obtainedfrom bacteria or archaebacteria, more particularly, from the organismHaloarcula marismortui. However, crystals may be obtained from ribosomesor ribosomal subunits from any organism, particularly from animals, moreparticularly from mammals, and even more particularly from humans.

[0040] In another preferred embodiment, the crystals diffract X-rays toa resolution of at least about 4.5 Å, more preferably to a resolution ofat least about 3.0 Å, and most preferably to a resolution of about 2.4 Åfor the determination of atomic co-ordinates of ribosomes or ribosomalsubunits. In another preferred embodiment, the crystals of the presentinvention may also include a ligand, for example, a protein synthesisinhibitor, for example, an antibiotic, (such as a macrolide antibiotic)complexed with, or bound to a ribosome or ribosomal subunit.

[0041] In another aspect, the invention provides crystals of 50Sribosomal subunits whose atomic structure is characterized by the atomicco-ordinates deposited at the Protein Data Bank ID: 1FFK or 1JJ2. Theinvention further provides phases computed from the co-ordinates of thedeposited co-ordinates and the uses of such phase information. In apreferred embodiment, the invention provides crystals of 50S ribosomalsubunits whose atomic structure is characterized by the atomicco-ordinates deposited at the Protein Data Bank ID: 1FFZ (largeribosomal subunit complexed with CCdA-p-Puro); or 1FG0 (large ribosomalsubunit complexed with a mini-helix analogue of aminoacyl-tRNA); as wellas those ribosomal subunits whose atomic structure is characterized bythe atomic coordinates listed in a file contained on Disk No. 3 of 3,specifically: large ribosomal subunit complexed with anisomycin (filename: anisomysin.pdb); large ribosomal subunit complexed withblasticidin (file name: blasticidin.pdb); large ribosomal subunitcomplexed with carbomycin (file name: carbomycin.pdb); large ribosomalsubunit complexed with tylosin (file name: tylosin.pdb); large ribosomalsubunit complexed with sparsomycin (file name: sparsomycin.pdb); largeribosomal subunit complexed with virginiarmycin (file name:virginiamycin.pdb); or large ribosomal subunit complexed with spiramycin(file name: spiramycin.pdb).

[0042] In another embodiment, the invention provides a method ofobtaining an electron density map of a ribosomal subunit of interestthat is only slightly different from the ribosomal subunit whosestructure has already been determined, for example, by X-raycrystallography. The method comprises the steps of: (a) producing acrystal of the ribosomal subunit of interest, wherein the crystal isisomorphous; (b) obtaining diffraction amplitudes of the crystalproduced in step (a); (c) combining the phases of the crystal of theribosomal subunit whose structure is already known with the diffractionamplitudes obtained in step (b) to produce a combined data set; and (d)obtaining an electron density map of the selected ribosomal subunitbased on the combined data set obtained in step (c).

[0043] In another embodiment, the invention further provides a method ofobtaining an electron density map of a ribosomal subunit of interestwhich is related to a ribosomal subunit whose structure is known. Themethod comprises the steps of: (a) producing a crystal of a ribosomalsubunit of interest, wherein the crystal crystallizes in a differentunit cell with different symmetry than the crystal of the ribosomalsubunit whose structure is known; (b) obtaining X-ray diffraction dataof the crystal of interest; (c) inserting the atomic co-ordinates of theknown ribosomal subunit into the unit cell of the crystal of interestand modeling the co-ordinates such that they would be capable ofproducing theoretical X-ray diffraction data that resembles the X-raydiffraction data obtained in step (b); (d) obtaining phases of thecrystal of interest from the modeled co-ordinates in step (c); and (e)obtaining an electron density map of the ribosomal subunit of interestfrom the X-ray diffraction data obtained in step (b) and the phasesobtained in step (d).

[0044] In addition, the invention provides a method of obtaining a modelof a ribosomal subunit of interest, where the ribosomal subunit ofinterest diverges significantly from but is still homologous to theribosomal subunit that was used to generate computed phases. The methodcomprises the steps of: (a) providing the atomic co-ordinates of theribosomal subunit whose structure is known; and (b) using homologymodeling to produce atomic co-ordinates of the ribosomal subunit ofinterest.

[0045] In another aspect, the invention provides a method of growing acrystal of a ribosome or a ribosomal subunit, as well as crystalsresulting from such a method. The method comprises the steps of: (a)isolating a ribosome or a ribosomal subunit; (b) precipitating theribosome or ribosomal subunit; (c) back-extracting the precipitatedribosome or ribosomal subunit to obtain a solution; (d) seeding theback-extracted solution; (e) growing a crystal of the ribosome orribosomal subunit from the seeded solution by vapor diffusion at roomtemperature; and (f) harvesting the crystal. Optionally, the method mayfurther comprise one or more of the following steps: (g) stabilizing thecrystal by gradual transfer into a solution containing high saltconcentration, for example, from about 1.2 M salt to about 1.7 M salt;(h) maintaining the crystal under such a high salt concentration; and(i) flash freezing the crystal.

[0046] In another aspect, the invention provides a method of obtainingX-ray diffraction data for a crystal of a ribosome or a ribosomalsubunit. The method comprises the steps of: (a) obtaining a crystal of aribosome or a ribosomal subunit, wherein the crystal has one or more ofthe following characteristics (1) an average thickness of greater than15 μm, and (2) untwinned; and (b) using X-ray crystallography to obtainX-ray diffraction data for the crystal of the ribosome or ribosomalsubunit. The present invention also discloses a method of obtaining anelectron density map of a ribosome or a ribosomal subunit comprisingusing the X-ray diffraction data described herein to obtain an electrondensity map of the ribosome or ribosomal subunit.

[0047] In another aspect, the invention provides a method of obtainingX-ray diffraction data for a complex of a ribosome and a ligand, forexample, a protein synthesis inhibitor, or a complex of a ribosomalsubunit and a ligand. The method comprises the steps of (a) obtaining acrystal of a ribosome or a ribosomal subunit, wherein the crystal hasone or more of the following characteristics: (1) an average thicknessof greater than 15 μm, and (2) untwinned; (b) diffusing a ligand intothe crystal and permitting the ligand to attach to the crystal so as toform a complex; and (c) using X-ray crystallography to obtain X-raydiffraction data for the complex. In an alternative aspect, theinvention provides a method of obtaining X-ray diffraction data for acomplex of a ribosome and a ligand, for example, a protein synthesisinhibitor or for a ribosomal subunit and a ligand. The method comprisesthe steps of: (a) obtaining a co-crystal for a complex of a ribosome anda ligand or for a complex of a ribosomal subunit and a ligand, whereinthe co-crystal has one or more of the following characteristics: (1) anaverage thickness of greater than 15 μm, and (2) untwinned; and (b)using X-ray crystallography to obtain X-ray diffraction data for thecomplex. In either method, the X-ray diffraction data can be used toproduce an electron density map for a complex of a ribosome and a ligandor for a complex of a ribosomal subunit and a ligand.

[0048] In a preferred embodiment, the invention provides a method oflocating the attachment of such a ligand to a ribosome or the attachmentof the ligand to a ribosomal subunit. The method comprises the steps of:(a) obtaining X-ray diffraction data for a ribosome or for a ribosomalsubunit; (b) obtaining X-ray diffraction data for a complex of aribosome and a ligand or for a complex of a ribosomal subunit and aligand; (c) subtracting the X-ray diffraction data obtained in step (a)from the X-ray diffraction data obtained in step (b) to obtain thedifference in the X-ray diffraction data; (d) obtaining phases thatcorrespond to X-ray diffraction data obtained in step (a) using one ormore of the techniques selected from the group consisting of MIR, MIRAS,SAD and computation from an existing atomic structure; (e) utilizing thephases obtained in step (d) and the difference in the X-ray diffractiondata obtained in step (c) to compute a difference Fourier image of theligand; and (f) locating the attachment of the ligand to a ribosome orthe attachment of the ligand to a ribosomal subunit based on thecomputations obtained in step (e).

[0049] In another embodiment, the invention provides an alternativemethod of obtaining a map of such a ligand attached to a ribosome or ofa ligand attached to a ribosomal subunit. The method comprises the stepsof: (a) obtaining X-ray diffraction data for a ribosome or for aribosomal subunit; (b) obtaining X-ray diffraction data for a complex ofa ribosome and a ligand or a complex of a ribosomal subunit and aligand; (c) obtaining phases that correspond to X-ray diffraction dataobtained in step (a) using one or more of the techniques selected fromthe group consisting of MIR, MIRAS, SAD and computation from an existingatomic structure; and (d) utilizing the phases obtained in step (c) andthe X-ray diffraction data obtained in step (b) to compute a map of theligand and the ribosome or of the ligand and the ribosomal subunit.

[0050] In another aspect, the invention provides a computer systemcomprising: (a) a memory having stored therein data indicative of atomicco-ordinates derived from an electron density map having a resolution ofat least about 4.5 Å, more preferably of at least about 3.0 Å, and mostpreferably of about 2.4 Å and defining a ribofunctional locus of a largesubunit of a ribosome; and (b) a processor in electrical communicationwith the memory, the processor comprising a program for generating athree-dimensional model representative of the ribofunctional locus. In apreferred embodiment, the computer system further comprises a device,for example, a computer monitor, or terminal for providing a visualrepresentation of the molecular model. In another preferred embodiment,the processor further comprises one or more programs to facilitaterational drug design.

[0051] In a preferred embodiment, the computer system further comprisesat least a portion of the atomic co-ordinates deposited at the ProteinData Bank under accession number PDB ID: 1FFK, 1FFZ, 1FG0, or 1JJ2. Inanother preferred embodiment, the atomic co-ordinates further define atleast a portion of a protein synthesis inhibitor, for example, anantibiotic, more specifically an antibiotic selected from the groupconsisting of anisomycin, blasticidin, carbomycin, sparsomycin,spiramycin, tylosin and virginiamycin, complexed with a ribofunctionallocus, for example, at least a portion of the atomic co-ordinatesrecorded on compact disk Disk No. 3 of 3, included herein.

[0052] In a preferred embodiment, the ribofunctional locus comprises atleast a portion of an active site in the ribosomal subunit, for example,at least a portion of one or more of: a peptidyl transferase site (aportion of which may be defined by a plurality of residues set forth inTable 5); an A-site (a portion of which may be defined by a plurality ofresidues set forth in Table 6); a P-site (a portion of which may bedefined by a plurality of residues set forth in Table 7); a polypeptideexit tunnel (a portion of which may be defined by a plurality ofresidues set forth in Table 8, Table 9 or Table 10); or an antibioticbinding domain (a portion of which may be defined by a plurality ofresidues set forth in Table 11, Table 12, Table 13, Table 14, Table 15,Table 16 or Table 17). Plurality of residues shall be considered toinclude at least 3 residues, preferably at least 5 residues, and morepreferably at least 10 residues. The ribofunctional locus may be definedby atoms of ribosomal RNA, one or more ribosomal proteins, or acombination of ribosomal RNA and one or more ribosomal proteins.

[0053] In another preferred embodiment, the atomic co-ordinates areproduced by molecular modeling. Using the atomic co-ordinates providedherein, the skilled artisan may generate models of any ribosome ofinterest using conventional techniques, for example, conventionalhomology modeling, and or molecular replacement techniques. In anotherembodiment, the atomic co-ordinates are produced by homology modelingusing at least a portion of the atomic co-ordinates deposited at theProtein Data Bank under accession number PDB ID: 1FFK, 1FFZ, 1FG0, or1JJ2, or the atomic co-ordinates included in compact disk Disk No. 3 of3. In another embodiment, the atomic co-ordinates are produced bymolecular replacement using at least a portion of the atomicco-ordinates deposited at the Protein Data Bank under accession numberPDB ID: 1FFK, 1FFZ, 1FG0, or 1JJ2, or the atomic co-ordinates includedin compact disk Disk No. 3 of 3.

[0054] In a preferred embodiment, the atomic co-ordinates defineresidues that are conserved between ribosomes or ribosomal subunits ofpathogens, for example, prokaryotic organisms, and, optionally but morepreferably, are also absent from ribosomes or ribosomal subunits of ahost organism, for example, a human. In another preferred embodiment,the atomic co-ordinates may define residues that are conserved betweenribosomes or ribosomal subunits of prokaryotic organisms, for example,bacteria, and, optionally but more preferably, are also absent fromribosomal subunits of eukaryotes, for example, a mammal, morepreferably, a human. This information can be used, for example, via theuse of one or more molecular models, to identify targets for rationaldrug design that may be exploited to develop new molecules, for example,protein synthesis inhibitors, that disrupt protein synthesis in apathogen, for example, a bacteria, but do not disrupt or otherwisesubstantially affect protein synthesis in a host organism, for example,a human.

[0055] In another aspect, the invention provides a variety of methodsfor designing, testing and refining new molecules via rational drugdesign. For example, the invention provides a method that comprises thesteps of: (a) providing a model, for example, a molecular model, havinga ribofunctional locus of a large subunit of a ribosome, wherein themodel is defined by the spatial arrangement of atoms derived from anelectron density map having a resolution of at least about 4.5 Å, morepreferably to at least about 3.0 Å, and most preferably to about 2.4 Å;and (b) using the model to identify a candidate molecule having asurface complementary to the ribofunctional locus. Preferably, thecandidate molecule stereochemically interfits and more preferably bindswith the ribofunctional locus of the large subunit of the ribosome.

[0056] In a preferred embodiment, the method comprises one or moreadditional steps of: producing the candidate molecule identified in sucha method; determining whether the candidate molecule, when produced,modulates (for example, induces or reduces) ribosomal activity;identifying a modified molecule; producing the modified molecule;determining whether the modified molecule, when produced, modulatesribosomal activity; and producing the modified molecule for use eitheralone or in combination with a pharmaceutically acceptable carrier. Thecandidate molecule and/or the modified molecule may be an antibiotic orantibiotic analogue, for example, a macrolide antibiotic or a macrolideanalogue.

[0057] In a preferred embodiment, the ribofunctional locus used in sucha method comprises at least a portion of an active site in the ribosomalsubunit. In another preferred embodiment, the ribofunctional locus isdefined by at least a portion of one or more of: a peptidyl transferasesite (a portion of which may be defined by a plurality of residues setforth in Table 5); an A-site (a portion of which may be defined by aplurality of residues set forth in Table 6); a P-site (a portion ofwhich may be defined by a plurality of residues set forth in Table 7); apolypeptide exit tunnel (a portion of which may be defined by aplurality of residues set forth in Table 8, Table 9 or Table 10); or anantibiotic binding domain (a portion of which may be defined by aplurality of residues set forth in Table 11, Table 12, Table 13, Table14, Table 15, Table 16 or Table 17). The ribofunctional locus may bedefined by atoms of ribosomal RNA, one or more ribosomal proteins, or acombination of ribosomal RNA and one or more ribosomal proteins.

[0058] In another preferred embodiment, the atomic co-ordinates are usedto produce a molecular model in an electronic form. The atomicco-ordinates preferably are produced by molecular modeling. In anotherembodiment, the atomic co-ordinates are produced by homology modelingusing at least a portion of the atomic co-ordinates deposited at theProtein Data Bank under accession number PDB ID: 1FFK, 1FFZ, 1FG0, or1JJ2, or the atomic co-ordinates included in compact disk Disk No. 3 of3. In another embodiment, the atomic co-ordinates are produced bymolecular replacement using at least a portion of the atomicco-ordinates deposited at the Protein Data Bank under accession numberPDB ID: 1FFK, 1FFZ, 1FG0, or 1JJ2, or the atomic co-ordinates includedin compact disk Disk No. 3 of 3.

[0059] In a preferred embodiment, the atomic co-ordinates may defineresidues that are conserved among ribosomes or ribosomal subunits ofpathogens, for example, prokaryotic organisms, and, optionally but morepreferably, are also absent in ribosomes or ribosomal subunits of a hostorganism, for example, a human. In another preferred embodiment, theatomic co-ordinates may define residues that are conserved betweenribosomes or ribosomal subunits of prokaryotic organisms, for example,bacteria, and, optionally but more preferably, are also absent fromribosomes or ribosomal subunits of eukaryotes, for example, a mammal,more preferably a human. This information can be used, for example, viathe use of one or more molecular models, to identify targets forrational drug design that may be exploited to develop new molecules, forexample, protein synthesis inhibitors, that disrupt protein synthesis ina pathogen, for example, a bacteria but do not disrupt or otherwisesubstantially affect protein synthesis in a host organism, for example,a human.

[0060] In a preferred embodiment, the invention provides a method ofobtaining a modified agent. The method comprises the steps of: (a)obtaining a crystal of a ribosome or of a ribosomal subunit; (b)obtaining the atomic co-ordinates of the crystal; (c) using the atomicco-ordinates and one or more molecular modeling techniques, for example,graphic molecular modeling and computational chemistry, to determine howto modify the interaction of an agent with a ribosome or ribosomalsubunit; and (d) modifying the agent based on the determinationsobtained in step (c) to produce a modified agent. Alternatively, themethod further comprises contacting the modified agent with a ribosomeor ribosomal subunit and detecting the interaction of the agent to theribosome or ribosomal subunit. The present invention also provides sucha modified agent (preferably a therapeutic agent), wherein the modifiedagent binds differently to a ribosome or ribosomal subunit than does theagent from which the modified agent was derived.

[0061] In another aspect, the invention provides new protein synthesisinhibitors that disrupt the function of a target ribosome. Theseinhibitors can be readily designed and tested as disclosed herein.

[0062] One type of protein synthesis inhibitor of the inventioncomprises: a first binding domain having a surface, for example, asolvent accessible surface, that mimics or duplicates a surface of aknown first molecule, for example, a first antibiotic, that binds with afirst contact site, for example, a first ribofunctional locus, in or ona large ribosomal subunit; and a second binding domain having a surface,for example, a solvent accessible surface, that mimics or duplicates asurface of a known second molecule, for example, a second antibiotic,that binds with a second contact site, for example, a secondribofunctional locus, in or on the ribosomal subunit. The first domainis attached to the second domain so as to permit both the first domainand the second domain to bind simultaneously with their respectivecontact sites within or on the ribosomal subunit so as to disruptprotein synthesis in a ribosomal subunit.

[0063] Another type of protein synthesis inhibitor is a synthetic,engineered molecule that comprises: a binding domain having a surface,for example, a solvent accessible surface, that mimics or duplicates asolvent accessible surface of a known molecule, for example, a firstknown antibiotic, which binds with a contact site, for example, aribofunctional locus in or on a ribosomal subunit; and an effectordomain attached to the binding domain which, upon binding of the bindingdomain with the contact site, occupies a space within or adjacent theribosomal subunit thereby to disrupt protein synthesis in the ribosomalsubunit.

[0064] The foregoing aspects and embodiments of the invention may bemore fully understood by reference to the following figures, detaileddescription and claims. Further advantages are evident from the drawings(provided in both grayscale and color).

BRIEF DESCRIPTION OF THE DRAWINGS

[0065] The patent or application file contains at least one drawingexecuted in color. Copies of this patent or patent applicationpublication with color drawing(s) will be provided by the Office uponrequest and payment of the necessary fee.

[0066] Color renditions similar to many of the following figures can befound, for example, in Ban et al. (2000) Science 289: 905-920; or Nissenet al. (2000) Science 289: 920-929.

[0067] The objects and features of the invention may be more fullyunderstood by reference to the drawings described below:

[0068] FIGS. 1(A)-(E) show the electron density from a 2.4 Å resolutionelectron density map. Specifically, FIG. 1(A) shows a stereo view of ajunction between 23S rRNA domains II, III, and IV. FIG. 1(B) shows theextended region of protein L2 interacting with surrounding RNA. FIG.1(C) shows in detail the L2 region with a bound Mg²⁺ ion. FIG. 1(D)shows in detail L2 with amino acid side chains. FIG. 1(E) shows helices94-97 from domain 6.

[0069]FIG. 2 shows the H. marismortui large ribosomal subunit in thecrown view. The subunit is shown in the crown view, with its L7/L12stalk to the right, its L1 stalk to the left, and its centralprotuberance (CP) up. In this view, the surface of the subunit thatinteracts with the small ribosomal subunit faces the reader. RNA isshown in gray in a space-filling rendering. The backbones of theproteins visible are rendered in gold. A transition state analogue boundto the peptidyl transferase site of the subunit is indicated in green.The particle is approximately 250 Å across.

[0070] FIGS. 3(A)-(B) show the secondary structure of the 23S rRNA fromH. marismortui. The secondary structure of this 23S rRNA is shown in astandardized format. FIG. 3(A) shows the 5′ half of the large subunitrRNA. FIG. 3(B) show the 3′ half of the large subunit rRNA. This diagramshows all the base pairings seen in the crystal structure of the largesubunit that are stabilized by at least two hydrogen bonds. Pairingsshown in red were predicted and are observed. Those shown in green werepredicted, but are not observed. Interactions shown in blue areobserved, but were not predicted. Bases shown in black do not appear tobe involved in pairing interactions. Sequences that cannot be visualizedin the 2.4 Å resolution electron density map are depicted in gray withthe secondary structures predicted for them.

[0071] FIGS. 4(A)-(L) show the tertiary structures of the RNA domains inthe H. marismortui large ribosomal subunit, its RNA as a whole, andschematics of its RNAs. Specifically, FIGS. 4(A) and 4(B) show the RNAstructure of the entire subunit. Domains are color coded as shown in theschematic of FIG. 5(C). FIG. 4(A) shows the particle in the crown view.FIG. 4(B) shows the image in FIG. 4(A) rotated 180° about an axisrunning vertically in the plane of the image. FIGS. 4(C) and 4(D) show aschematic diagram of 23S rRNA and the secondary structure of 5S rRNA.FIG. 4(C) shows a schematic diagram of 23S rRNA secondary structure ofFIG. 3 with helices numbered according to Leffers et al. ((1987) J. Mol.Biol. 195: 43-61), and the domains of the molecule are indicated bycolor shading. FIG. 4(D) shows the secondary structure of 5S rRNA fromH. marismortui. Thick lines joining bases represent Watson-Crickpairing. Bases joined by a lower case “o” indicate non-Watson-Crickpairing. Bases joined by thin lines interact via a single hydrogen bond.Bases shown in black are not paired. Bases shown in red arephylogenetically predicted pairing that have now been confirmed(Symanski et al. (1998) Nucl. Acids Res. 26: 156-159). Pairs shown inblue are observed, but were not predicted, and pairs shown in green werepredicted but are not observed. FIGS. 4(E) through 4(L) show stereoviews of the RNA domains in the 23S rRNA and of 5S rRNA. Each domain iscolor-coded from its 5′ end to its 3′ end to facilitate the viewerfollowing its trajectory in three-dimensions. The surfaces where themost important inter-domain interactions occur are shown in mono to theright of the stereo views. FIG. 4(E) shows domain I; FIG. 4(F) showsdomain II; FIG. 4(G) shows domain III; FIG. 4(H) shows domain IV; FIG.4(I) shows domain V, crown view; FIG. 4(J) shows domain V, back view;FIG. 4(K) shows domain VI; and FIG. 4(L) shows 5S rRNA.

[0072] FIGS. 5(A)-(C) show conservations and expansions in the 23S rRNAof H. marismortui. The generality of the RNA in these images is gray.Sequences that are found to be >95% conserved across the threephylogenetic kingdoms are shown in red. Sequences where expansion in thebasic 23S structure is permitted are shown in green (Gutell et al.(2000) supra). Specifically, FIG. 5(A) shows the particle rotated withrespect to the crown view so that its active site cleft can be seen.FIG. 5(B) shows the crown view. FIG. 5(C) shows the back view of theparticle, i.e., the crown view rotated 180° about its vertical axis.

[0073] FIGS. 6(A)-(I) show structures of some large subunit ribosomalproteins that have non-globular extensions. Only the backbones of theproteins are shown. The globular domains of these proteins are shown ingreen, and their non-globular extensions are depicted in red. Thepositions of the zinc ions in L44e and L37e are also indicated. FIG.6(A) shows L2; FIG. 6(B) shows L3; FIG. 6(C) shows L39; FIG. 6(D) showsL4; FIG. 6(E) shows L15; FIG. 6(F) shows L21e; FIG. 6(G) shows L44e;FIG. 6(H) shows L37e; FIG. 6(I) shows L19;

[0074] FIGS. 7(A)-(C) show proteins that appear on the surface of thelarge ribosomal subunit. The RNA of the subunit is shown in gray, as inFIG. 2, and protein backbones are shown in gold. Specifically, FIG. 7(A)shows the subunit in the crown view of the subunit. FIG. 7(B) shows theback side of the subunit in the crown view orientation. FIG. 7(C) showsthe bottom view; the end of the peptide tunnel appears in the center ofthis image. The proteins visible in each image are identified in thesmall images at the lower left corner of the Figure.

[0075] FIGS. 8(A)-(F) show the protein distribution and protein-RNAinteractions in the large ribosomal subunit. Specifically, FIG. 8(A)shows the structures of proteins in the neighborhood of the end of thepeptide tunnel and how they relate to the RNA sequences with which theyinteract. Protein L22 extends a long P hairpin extension inside the 23SrRNA. L24 has a similar extension but the entire protein is on thesurface of the particle. L39 is the only protein in the subunit thatlacks tertiary structure, while L37e has both NH₂ and C00H terminalextensions. L19 is unique in having two globular domains on the surfaceof the subunit connected by an extended sequence that weaves through theRNA. The end of L39 (green) actually enters the tunnel, while L37e (red)is entirely surrounded by RNA. FIG. 8(B) shows the non-globularextensions of L2 and L3 reaching through the mass of 23S rRNA towardsthe peptidyl transferase site, which is marked by a CCdA-p-puromycinmolecule. FIG. 8(C) shows L22 interacting with portions of all six ofthe domains of 23S rRNA. FIG. 8(D) shows a schematic of 23S rRNA showingthe locations of the sequences that make at least van der Waals contactwith protein (red). FIG. 8(E) shows a stereo view of the proteins of thelarge ribosomal subunit with all the RNA stripped away. Proteins arecolor red as an aid to visualization only. FIG. 8(F) shows a crosssection of the subunit in the area of the tunnel exit. Protein L22 isshown as ribbons in red, and the β hairpin loop where mutations confererythromycin resistance is shown in orange. Atoms on the surface areshown in gray, protein atoms are shown in green, and atoms at the sliceinterface are shown in blue.

[0076] FIGS. 9(A)-(C) show chemical structures of ribosome peptidyltransferase substrates and analogues. Specifically, FIG. 9(A) shows thetetrahedral carbon intermediate produced during peptide bond formation;the tetrahedral carbon is indicated by an arrow. FIG. 9(B) shows thetransition state analogue formed by coupling the 3′ OH of CCdA to theamino group of the 0-methyl tyrosine residue of puromycin via aphosphate group, CCdA-p-Puro (Welch et al. (1995) supra). FIG. 9(C)shows an amino-N-acylated mini helix constructed to target the A-site.The oligonucleotide sequence 5′ phosphateCCGGCGGGCUGGUUCAAACCGGCCCGCCGGACC 3′ (SEQ ID NO: 1) puromycin shouldform 12 base pairs. The construct was based on a mini helix which is asuitable substrate for amino-acylation by Tyr-tRNA synthetase. The 3′ OHof its terminal C is coupled to the 5′ OH of the N6-dimethyl A moiety ofpuromycin by a phosphodiester bond.

[0077] FIGS. 10(A)-(C) show experimentally phased electron density mapsof the substrate analogue complexes at 3.2 Å resolution, with modelssuperimposed (oxygen, red; phosphorus, purple; nitrogen, blue; andcarbon, green for rRNA and yellow for substrate). Specifically, FIG.10(A) shows an F_(o)(complex)-F_(o)(parent) difference electron densitymap with a skeletal model of CCdA-p-Puro superimposed. FIG. 10(B) showsa 2F_(o)(complex)-F_(o)parent) electron density map of the CCdA-p-Puroin the active site region with the structures of the ribosome andinhibitor superimposed showing the proximity of the N3 of A2486 (2451)to the phosphate, non-bridging oxygen in this complex. FIG. 10(C) showsan F_(o)(complex)-F_(o)(parent) differences electron density map of thetRNA acceptor stem analogue with a skeletal model of CCpurosuperimposed. There is density only for the ribose and phosphate of C74and none for the rest of the RNA hairpin.

[0078] FIGS. 11(A) and (B) show a combined model of the CCA portion ofthe mini helix bound to the A-site and CCdA-p-Puro bound to the A- andP-sites, color coded as in FIG. 2. Specifically, FIG. 11(A) shows thebase-pairing interactions between the P-site C74 and C75 and the P loopof 23S rRNA on the left and the A-site C75 with the A loop of 23S rRNAon the right. The catalytic A2486 is near the phosphate oxygen (P) thatis the analogue of the tetrahedral intermediate oxyanion. FIG. 11(B)shows A2637 (in all blue) lying between the two CCA's and A2486 (green)whose N3 approaches a non-bridging phosphate oxygen. The N1 atoms of theA76 bases from the A- and P-site tRNAs are making nearly identicalinteractions with a ribose 2′ OH in both the A- and P-loops,respectively, and an approximate 2-fold axis relates these residues.

[0079]FIG. 12 shows a space filling model of the 23S and 5S rRNA, theproteins and the CCdA-p-Puro inhibitor viewed down the active site cleftin a rotated “crown view.” The bases are white and the sugar phosphatebackbones are yellow. The inhibitor is shown in red and the numberedproteins are shown in blue. The L1 and L11 proteins positioned at lowerresolution are in blue backbone. The central protuberance is labeled CP.

[0080]FIG. 13(A) shows a stereo view diagram of the three-dimensionaldistribution of the residues comprising the loops A and P and thepeptidyl transferase loop. FIG. 13(B) shows a stereo view of the centralloop in domain V from the direction of the tunnel. The residues arecolor coded based on mutations which confer antibiotic resistance. FIG.13(C) shows domain V active site with its central loop shown as thesecondary structure.

[0081] FIGS. 14(A) and (B) show the closest approach of polypeptides tothe peptidyl transferase active site marked by a ball and stickrepresentation of the Yarus inhibitor, CCdA-p-Puro. Specifically, FIG.14(A) shows a coil representation of domain V RNA backbone in red andbases in gray and a ribbon backbone representation of all thirteenproteins that interact with it. FIG. 14(B) shows a close-up view of theactive site with the RNA removed. The phosphate of the Yarus analogueand the proteins whose extensions are closest to the inhibitor are shownin ribbon with their closest side-chains in all atom representation. Thedistances in Å between the closest protein atoms and the phosphorousanalogue of the tetrahedral carbon (pink) are shown, as is a modeledpeptide (pink).

[0082]FIG. 15 shows conserved nucleotides in the peptidyl transferaseregion that binds CCdA-p-Puro A space filling representation of theactive site region with the Yarus inhibitor viewed down the active sitecleft. All atoms belonging to 23S rRNA nucleotides that are 95%conserved in all three kingdoms (Gutell et al. (2000) supra) are coloredred and all other nucleotides are white; the inhibitor is colored blue.

[0083] FIGS. 16(A)-(C) show the catalytic apparatus of the peptidyltransferase active site. Specifically, FIG. 16(A) shows stereo view of aportion of the experimental 2.4 Å resolution electron density map (Banet al. (2000) Science 289: 905-920) of the large subunit in the regionof the catalytic site in stereo. The structure the RNA involved ininteractions with A2486 is superimposed. Residues G2102 (2061) and G2482(2447) are hydrogen bonded to the N6 of A2486 (2451) and G2482 whichinteracts with a neighboring phosphate group. FIG. 16(B) shows askeletal representation with dashed hydrogen-bonds showing G2482, G2102,A2486 and the buried phosphate that is proposed to result in a chargerelay through G2482 to the N3 of A2486. FIG. 16(C) shows the normal andrarer imine tautomeric forms of G2482 and A2486 that are proposed to bestabilized by the buried phosphate of residue 2485.

[0084] FIGS. 17(A)-(C) show the proposed mechanism of peptide synthesiscatalyzed by the ribosome. Specifically, FIG. 17(A) shows the N3 ofA2486 abstracting a proton from the NH₂ group as the latter attacks thecarbonyl carbon of the peptidyl-tRNA. FIG. 17(B) shows a protonated N3stabilizing the tetrahedral carbon intermediate by hydrogen bonding tothe oxyanion. FIG. 17(C) shows the proton transferred from the N3 to thepeptidyl tRNA 3′OH as the newly formed peptide deacylates.

[0085] FIGS. 18(A) and (B) show space filling representations of the 50Sribosomal subunit with the 3 tRNA molecules, in the same relativeorientation that they are found in the 70S ribosome structure by Nollerand colleagues docked onto the CCA's bound in the A-Site and P-Site.Specifically, FIG. 18(A), shown on the left-hand side, shows the wholesubunit in rotated crown view with the rRNA in yellow, proteins in pinkand tRNAs in orange. FIG. 18(B), shown on the right-hand side, shows aclose-up view showing the numbered proteins are in pink and the rRNA inblue. A backbone ribbon representation of the A-, P-, and E-sites areshown in yellow, red and white, respectively.

[0086] FIGS. 19(A)-(F) show the polypeptide exit tunnel. Specifically,FIG. 19(A) shows the subunit cut in half, roughly bisecting its centralprotuberance and its peptide tunnel along the entire length. The twohalves have been opened like the pages of a book. All ribosome atoms areshown in CPK representation, with all RNA atoms that do not contactsolvent shown in white and all protein atoms that do not contact solventshown in green. Surface atoms of both protein and RNA are color-codedwith carbon in yellow, oxygen in red, and nitrogen in blue. A possibletrajectory for a polypeptide passing through the tunnel is shown as awhite ribbon. The peptidyl transferase site (PT) is also shown. FIG.19(B) shows detail of the polypeptide exit tunnel with the distributionof polar and non-polar groups, with atoms colored as in FIG. 19(A), theconstriction in the tunnel formed by proteins L22 and L4 (green patchesclose to PT), and the relatively wide exit of the tunnel. A modeledpolypeptide is in white. FIG. 19(C) shows the tunnel surface withbackbone atoms of the RNA color coded by domain: domain I (white), II(light blue), III (gold), IV (green), V (orange), 5S (pink) and proteinsare blue. The peptidyl transferase center (PTC) is shown. FIG. 19(D) isa space filling representation of the large subunit surface at thetunnel exit showing the arrangement of proteins, some of which mightplay roles in protein secretion. The RNA is in white (bases) and yellow(backbone) and the numbered proteins are blue. A modeled polypeptide isexiting the tunnel in red. FIG. 19(E) shows a close-up view of the halfof the exit tunnel showing the relationship of the peptidyl transferasecenter (PTC) to proteins L4 (yellow) and L22 (blue). The Yarus inhibitorand a modeled peptide are purple and the 23S rRNA is in red and white.FIG. 19(F) shows a secondary structure schematic of 23S rRNA identifyingthe sequences that contact the tunnel in red.

[0087]FIG. 20 is a picture showing the spatial relationship between theantibiotic anisomycin bound to a large ribosomal subunit.

[0088]FIG. 21 is a picture showing the spatial relationship between theantibiotic blasticidin bound to a large ribosomal subunit.

[0089]FIG. 22 is a picture showing the spatial relationship between theantibiotics carbomycin and tylosin bound to a large ribosomal subunit.

[0090]FIG. 23 is a picture showing the spatial relationship between theantibiotic sparsomycin bound to a large ribosomal subunit.

[0091]FIG. 24 is a picture showing the spatial relationship between theantibiotics virginiamycin (streptogramin A) and carbomycin bound to alarge ribosomal subunit.

[0092]FIG. 25 is a picture showing the spatial relationship of certainantibiotics, namely, anisomycin, blasticidin, carbomycin, andvirginiamycin, bound to a large ribosomal subunit. The locations of thebound antibiotics are shown relative to the ribosomal A-site, P-site,and polypeptide exit tunnel.

[0093] FIGS. 26(A)-(C) are pictures showing a peptidyl transferase sitedisposed within a large ribosomal subunit. FIG. 26A shows a boundtylosin molecule, and identifies a disaccharide binding pocket and twocavities denoted “cavity 1” and “cavity 2.” FIGS. 26(B) and (C) areprovided on the left hand side to orient the reader to the locations ofthe peptidyl transferase site (PT) and polypeptide exit tunnel in thelarge ribosomal subunit.

[0094]FIG. 27 is a schematic representation of a computer system usefulin molecular modeling a ribosomal subunit and/or for performing rationaldrug design.

[0095]FIG. 28 is a schematic representation of certain potential drugtarget sites in a large ribosomal subunit.

[0096] FIGS. 29(A)-(D) are pictures showing the residues within the wallof the polypeptide exit tunnel that are conserved (red) or non-conserved(blue) between E. coli and rat. The ribosomal subunit has been sliceddown the polypeptide exit tunnel with one half of the polypeptide exittunnel shown in FIG. 29(A), and the other half of the polypeptide exittunnel is shown in FIG. 29(B). FIG. 29(C) is provided to orient thereader to show the location of the portion of the ribosomal subunitshown in FIG. 29(A) relative to the ribosomal subunit as a whole. FIG.29(D) is provided to orient the reader to show the location of theportion of the ribosomal subunit shown in FIG. 29(B) relative to thelarge ribosomal subunit as a whole.

DETAILED DESCRIPTION OF THE INVENTION I. Definitions

[0097] As used herein, the term “active site” refers to regions on aribosome or ribosomal subunit that are directly involved in proteinsynthesis, e.g., the peptidyl transferase site, the elongation factorbinding site, and other similar sites.

[0098] As used herein, the terms “agent” and “ligand” are usedsynonymously and refer to any atom, molecule, or chemical group whichbinds with a ribosome, ribosomal subunit or ribosome fragment. Thus,ligands include, but are not limited to, a single heavy atom, anantibiotic, a tRNA, a peptidyl tRNA, an aminoacyl tRNA, or a signalrecognition particle (“SRP”).

[0099] As used herein, “archaebacteria” refers to the kingdom ofmonerans that includes methane producers, sulfur-dependent species, andmany species that tolerate very salty or hot environments.

[0100] As used herein, the term “A-site” refers to the locus occupied byan aminoacyl-tRNA molecule immediately prior to its participation in thepeptide-bond forming reaction.

[0101] As used herein, the term “asymmetric unit” refers to a minimalset of atomic co-ordinates that when operated upon by the symmetryoperations of a crystal will regenerate the entire crystal.

[0102] As used herein, “at least a portion of” or “at least a portion ofthe three-dimensional structure of” is understood to mean a portion ofthe three-dimensional structure of a ribosome or ribosomal subunit,including charge distribution and hydrophilicity/hydrophobicitycharacteristics, formed by at least three, more preferably at leastthree to ten, and most preferably at least ten contiguous amino acidand/or nucleotide residues of the ribosome or ribosomal subunit. Thecontiguous residues forming such a portion may be residues which form acontiguous portion of the primary sequence of a ribosomal RNA orribosomal protein, residues which form a contiguous portion of thethree-dimensional structure of the ribosome or ribosomal subunit, or acombination thereof. Thus, the residues forming a portion of thethree-dimensional structure need not be contiguous in the primarysequence but, rather, must be contiguous in space. As used herein, theresidues forming “a portion of the three-dimensional structure of” aribosome or ribosomal subunit, form a contiguous three-dimensional shapein which each atom or functional group forming the portion of the shapeis separated from the nearest atom or functional group forming theportion of the shape by no more than 40 Å, preferably by no more than 20Å, more preferably by no more than 5-10 Å, and most preferably by nomore than 1-5 Å.

[0103] As used herein, the term “atomic co-ordinates” or “structureco-ordinates” refers to mathematical co-ordinates (represented as “X,”“Y” and “Z” values) that describe the positions of atoms in a crystal ofa ribosome or ribosomal subunit. The diffraction data obtained from thecrystals are used to calculate an electron density map of the repeatingunit of the crystal. The electron density maps are used to establish thepositions of the individual atoms within a single ribosomal subunit.Those of skill in the art understand that a set of structureco-ordinates determined by X-ray crystallography is not without standarderror. For the purpose of this invention, any set of structureco-ordinates for a ribosome or ribosomal subunit from any source has aroot mean square deviation of non-hydrogen atoms of less than 0.75 Åwhen superimposed on the non-hydrogen atom positions of the said atomicco-ordinates deposited at the Research Collaboratory for StructuralBioinformatics (RCSB) Protein Data Bank (PDB) (Berman et al. (2000)Nucleic Acids Research 28, 235-242; http://www.rcsb.org/pdb/) with theaccession numbers PDB ID: 1FFK; PDB ID: 1FFZ; PDB ID: 1FG0; or PDB ID:1JJ2, the disclosure of each of the foregoing of which is incorporatedherein by reference in its entirety.

[0104] In the list of atomic co-ordinates deposited at the RCSB ProteinData Bank or included herein as files recorded on the compact disks, theterm “atomic co-ordinate” or structure co-ordinates refer to themeasured position of an atom in the structure in Protein Data Bank (PDB)format, including X, Y, Z and B, for each. The term “atom type” refersto the element whose co-ordinates are measured. The first letter in thecolumn defines the element. The term “X”, “Y”, “Z” refers to thecrystallographically defined atomic position of the element measuredwith respect to the chosen crystallographic origin. The term “B” refersto a thermal factor that measures the mean variation of an atom'sposition with respect to its average position.

[0105] Reference is made to the sets of atomic co-ordinates and relatedtables included with this specification and submitted on compact disk(six total compact disks including three original compact disks, and aduplicative copy of each of the original compact disks), all of theforegoing of which are incorporated by reference herein. Disk No. 1 of 3contains eight files; Disk No. 2 of 3 contains four files; and Disk No.3 of 3 contains nine files. Disk No. 1 of 3 contains the filesidentified as PDBlFFK.DOC and PDB1FFK.ENT which represent files ofco-ordinates defining the large ribosomal subunit; PDB 1FFZ.DOC and PDB1 FFZ.ENT which represent files of the co-co-ordinates defining thelarge ribosomal subunit—CCdA-p-Puro complex; and PDB1FGO.DOC andPDB1FGO.ENT which represent files of the co-ordinates defining the largeribosomal subunit—aa-tRNA analogue complex. Disk No. 2 of 3 containsfiles identified as 1JJ2.RTF and 1JJ2.TXT which represent files of theco-ordinates defining the completely refined large ribosomal subunit.Disk No. 3 of 3 contains the files identified as anisomycin.pdb,blasticidin.pdb, carbomycin.pdb, sparsomycin.pdb, spiramycin.pdb,tylosin.pdb and virginiamycin.pdb which represent files of theco-ordinates defining the large ribosomal subunit bound to anisomycin,blasticidin, carbomycin, sparsomycin, spiramycin, tylosin, andvirginiamycin, respectively.

[0106] As will be apparent to those of ordinary skill in the art, theatomic structures presented herein are independent of their orientation,and that the atomic co-ordinates identified herein merely represent onepossible orientation of a particular large ribosomal subunit. It isapparent, therefore, that the atomic co-ordinates identified herein maybe mathematically rotated, translated, scaled, or a combination thereof,without changing the relative positions of atoms or features of therespective structure. Such mathematical manipulations are intended to beembraced herein.

[0107] As used herein, the terms “atomic co-ordinates derived from” and“atoms derived from” refers to atomic co-ordinates or atoms derived,either directly or indirectly, from an electron density map. It isunderstood that atomic co-ordinates or atoms derived “directly” from anelectron density map refers to atomic co-ordinates or atoms that areidentified from and/or fitted to an electron density map by usingconventional crystallographic and/or molecular modeling techniques andthus can be considered to be primary atomic co-ordinates or atoms. It isunderstood that atomic co-ordinates or atoms derived “indirectly” froman electron density map refers to atomic co-ordinates or atoms that arederived from and thus are derivatives or transforms of the primaryatomic co-ordinates or atoms and thus can be considered to be secondaryatomic co-ordinates or atoms. The secondary atomic co-ordinates or atomsmay be generated from the primary atomic co-ordinates or atoms by usingconventional molecular modeling techniques. By way of a non limitingexample, the atomic co-ordinates for the H. marismortui large ribosomalsubunit as described hereinbelow are considered to be primaryco-ordinates, whereas the atomic co-ordinates of a mammalian largeribosomal subunit which can be derived from H. marismortui atomicco-ordinates by molecular modeling, including, for example, homologymodeling and/or molecular replacement, are considered to be secondaryco-ordinates. Both types of atomic co-ordinates and atoms are consideredto be embraced by the invention.

[0108] As used herein the terms “bind,” “binding,” “bound,” “bond,” or“bonded,” when used in reference to the association of atoms, molecules,or chemical groups, refer to any physical contact or association of twoor more atoms, molecules, or chemical groups (e.g., the binding of aligand with a ribosomal subunit refers to the physical contact betweenthe ligand and the ribosomal subunit). Such contacts and associationsinclude covalent and non-covalent types of interactions.

[0109] As used herein, the terms “complex” or “complexed” refer to theassembly of two or more molecules to yield a higher order structure,such as, a 50S ribosomal subunit bound to a ligand.

[0110] As used herein, the term “computational chemistry” refers tocalculations of the physical and chemical properties of the molecules.

[0111] As used herein, the term “conjugated system” refers to more thantwo double bonds that are positioned spatially so that their electronsare completely delocalized with the entire system. Aromatic residuescontain conjugated double bond systems.

[0112] As used herein, the terms “covalent bond” or “valence bond” referto a chemical bond between two atoms in a molecule created by thesharing of electrons, usually in pairs, by the bonded atoms.

[0113] As used herein, the term “crystal” refers to anythree-dimensional ordered array of molecules that diffracts X-rays.

[0114] As used herein, the term “crystallographic origin” refers to areference point in the unit cell with respect to the crystallographicsymmetry operation.

[0115] As used herein, the term “elongation factor binding domain”refers to the region of the ribosome that interacts directly withelongation factors, including, for example, the elongation factors,EF-Tu and EF-G.

[0116] As used herein, the term “E-site” refers to the locus occupied bya deacylated tRNA molecule as it leaves the ribosome following itsparticipation in peptide-bond formation.

[0117] As used herein, the term “heavy atom derivatization” refers tothe method of producing a chemically modified form, also known as a“heavy atom derivative”, of a crystal of the ribosome and the ribosomalsubunit and its complexes. In practice, a crystal is soaked in asolution containing heavy metal atom salts, or organometallic compounds,e.g., mercury chlorides, ethyl-mercury phosphate, osmium pentamine, oriridium pentamine, which can diffuse through the crystal and bind to theribosome or ribosomal subunit. The location(s) of the bound heavy metalatom(s) can be determined by X-ray diffraction analysis of the soakedcrystal. This information, in turn, is used to generate the phaseinformation used to construct three-dimensional structure of the complex(Blundell et al. (1976) supra).

[0118] As used herein, the term “homologue” is understood to mean anyone or combination of (i) any protein isolated or isolatable from aribosome or a ribosomal subunit (i.e., a ribosomal protein), (ii) anynucleic acid sequence isolated or isolatable from a ribosome orribosomal subunit (i.e., a ribosomal RNA), (iii) any protein having atleast 25 % sequence identity to a ribosomal protein isolated from E.coli or Rattus norvegicus as determined using the computer program“BLAST” version number 2.1.1 implementing all default parameters, or(iv) any nucleic acid having at least 30% sequence identity to aribosomal RNA isolated from E. coli or Rattus norvegicus as determinedusing the computer program “BLAST” version number 2.1.1 implementing alldefault parameters. “BLAST” version number 2.1.1 is available andaccessible via the world wide web at http:/www/ncbi.nlm.nih.gov/BLAST/or can be run locally as a fully executable program on a standalonecomputer.

[0119] As used herein, the term “homology modeling” refers to thepractice of deriving models for three-dimensional structures ofmacromolecules from existing three-dimensional structures for theirhomologues. Homology models are obtained using computer programs thatmake it possible to alter the identity of residues at positions wherethe sequence of the molecule of interest is not the same as that of themolecule of known structure.

[0120] As used herein, the term “hydrogen bond” refers to twoelectronegative atoms (either O or N), which share a hydrogen that iscovalently bonded to only one atom, while interacting with the other.

[0121] As used herein, the term “hydrophobic interaction” refers tointeractions made by two hydrophobic residues.

[0122] As referred to herein, ribosomal proteins are designated “LX” or“SX”, where L stands for “large subunit; S stands for “small subunit”;and X in either case is an integer.

[0123] As used herein, the term “MIR” refers to multiple isomorphousreplacement, a technique used for deriving phase information fromcrystals treated with heavy atom compounds.

[0124] As used herein, the term “molecular graphics” refers tothree-dimensional representations of atoms, preferably on a computerscreen.

[0125] As used herein, the terms “molecular model” or “molecularstructure” refer to the three-dimensional arrangement of atoms within aparticular object (e.g, the three-dimensional structure of the atomsthat comprise a ribosome or ribosomal subunit, and the atoms thatcomprise a ligand that interacts with a ribosome or ribosomal subunit,particularly with a large ribosomal subunit, more particularly with a50S ribosomal subunit).

[0126] As used herein, the term “molecular modeling” refers to a methodor procedure that can be performed with or without a computer to makeone or more models, and, optionally, to make predictions about structureactivity relationships of ligands. The methods used in molecularmodeling range from molecular graphics to computational chemistry.

[0127] As used herein, the term “molecular replacement” refers to amethod that involves generating a model of a ribosome or ribosomalsubunit whose atomic co-ordinates are unknown, by orienting andpositioning the atomic co-ordinates described in the present inventionin the unit cell of the crystals of the unknown ribosome so as best toaccount for the observed diffraction pattern of the unknown crystal.Phases can then be calculated from this model and combined with theobserved amplitudes to give the atomic co-ordinates of the unknownribosome or ribosomal subunit. This type of method is described, forexample, in The Molecular Replacement Method, (Rossmann, M. G., ed.),Gordon & Breach, New York, (1972).

[0128] As used herein, “noncovalent bond” refers to an interactionbetween atoms and/or molecules that does not involve the formation of acovalent bond between them.

[0129] As used herein, the term “peptidyl transferase site” refers tothe locus in the large ribosomal subunit where peptide bonds aresynthesized.

[0130] As used herein, the term “polypeptide exit tunnel” refers to thechannel that passes through the large ribosomal subunit from thepeptidyl transferase site to the exterior of the ribosome through whichnewly synthesized polypeptides pass.

[0131] As used herein, the term “protein synthesis inhibitor” refers toany molecule that can reduce, inhibit or otherwise disrupt protein orpolypeptide synthesis in a ribosome.

[0132] As used herein, the term “P-site” refers to the locus occupied bya peptidyl-tRNA at the time it participates in the peptide-bond formingreaction.

[0133] As used herein, the term “ribofunctional locus” refers to aregion of the ribosome or ribosomal subunit that participates, eitheractively or passively, in protein or polypeptide synthesis within theribosome or ribosomal subunit and/or export or translocation of aprotein or polypeptide out of a ribosome. The ribofunctional locus caninclude, for example, a portion of a peptidyl transferase site, anA-site, a P-site, an E-site, an elongation factor binding domain, apolypeptide exit tunnel, and a signal recognition particle (SRP) bindingdomain. It is understood that the ribofunctional locus will not onlyhave a certain topology but also a particular surface chemistry definedby atoms that, for example, participate in hydrogen bonding (forexample, proton donors and/or acceptors), have specific electrostaticproperties and/or hydrophilic or hydrophobic character.

[0134] As used herein, the term “ribosomal subunit” refers to one of thetwo subunits of the ribosome that can function independently during theinitiation phase of protein synthesis but which both together constitutea ribosome. For example, a prokaryotic ribosome comprises a 50S subunit(large subunit) and a 30S subunit (a small subunit).

[0135] As used herein, the term “ribosome” refers to a complexcomprising a large ribosomal subunit and small ribosomal subunit.

[0136] As used herein, the term “signal recognition particle bindingdomain” refers to the portion of the ribosome that interacts directlywith the signal recognition particle.

[0137] As used herein, the term “space group” refers to the arrangementof symmetry elements of a crystal.

[0138] As used herein, the term “symmetry operation” refers to anoperation in the given space group that places the atoms in oneasymmetric unit on the corresponding atoms in another asymmetric unit.

[0139] As used herein, the term “twinned” refers to a single macroscopiccrystal that contains microscopic domains of the same symmetry thatdiffer significantly in orientation in such a way that the diffractionpatterns of all are superimposed. In a twinned crystal the mosaicblocks, or domains, are orientated so that some point in one directionand others point in a second, distinctly different direction, and thedirections are such that the diffraction pattern generated by one groupof blocks falls exactly on top of the diffraction pattern of the othergroup.

[0140] As used herein, the term “untwinned” refers to a crystal cell thedomains of which are aligned. The domains are also known as the “mosaicblocks.” Most crystals diffract as though they were assemblies of mosaicblocks. One can think of them as small, perfectly ordered regions withinthe larger crystal, which, overall, is not so well ordered. Each blockhas the same symmetry and unit cell packing as all the others.

[0141] As used herein, the term “unit cell” refers to a basicparallelepiped shaped block. The entire volume of crystal may beconstructed by regular assembly of such blocks. Each unit cell comprisesa complete representation of the unit of pattern, the repetition ofwhich builds up the crystal.

II. Structure and Use of the Large Ribosomal Subunit

[0142] A. Atomic Structure of the Large Ribosomal Subunit at 2.4 ÅResolution, Initial Refinement

[0143] The present invention is based, in part, on the development of anovel method for preparing crystals of ribosomes. The novel methodprovides crystals of the 50S ribosomal subunit that are much thickerthan those available earlier and that can diffract X-rays to aresolution of about 2.4 Å. The method eliminates the twinning ofcrystals that obstructed progress in determining the crystal structureof the 50S ribosomal subunit from H. marismortui for many years. Themethod of preparing the crystals of the 50S ribosomal subunit isdiscussed below.

[0144] The present invention is also based, in part, on the atomicstructure of the crystal of the 50S ribosomal subunit from H.marismortui that has been derived from a 2.4 Å resolution electrondensity map that was experimentally phased using heavy atom derivatives.The atomic co-ordinates defining the large ribosomal unit were depositedon Jul. 10, 2000, at Research Collaboratory for StructuralBioinformatics (RCSB) Protein Data Bank (PDB) (Berman et al. (2000)Nucleic Acid Research 28, 235-242; http://www.rcsb.org/pdb/) withaccession number PDB1D: 1FFK.

[0145] Moreover, the present invention is based, in part, on thederivation from the atomic co-ordinates of the following model which isbriefly summarized here and discussed in detail in the followingsections of the specification. This model includes 2,811 of the 2,923nucleotides of 23S rRNA, all 122 nucleotides of its 5S rRNA, andstructures for the 27 proteins that are well-ordered in the subunit.

[0146] The secondary structures of both 5S and 23S rRNA are remarkablyclose to those deduced for them by phylogenetic comparison. Thesecondary structure of the 23S rRNA divides it into 6 large domains,each of which has a highly asymmetric tertiary structure. Theirregularities of their shapes notwithstanding, the domains fit togetherin an interlocking manner to yield a compact mass of RNA that is almostisometric. The proteins are dispersed throughout the structure,concentrated largely on its surface, but they are much less abundant inthe regions of the subunit that are of primary functional significanceto protein syntheses—the 30S subunit interface, the binding regions fortRNA and the peptidyl transferase active site. The most surprisingfeature of many of these proteins are the extended, irregular structuresof their loops and termini, which penetrate between RNA helices. Theprimary role of most of the proteins in the subunit appears to bestabilization of the three-dimensional structure of its rRNA.

[0147] 1. Preparation of the Crystal for the 50S Ribosomal Subunit andStructure Determination.

[0148] Several experimental approaches were used to extend theresolution of the electron density maps of the H. marismortui 50Sribosomal subunit from 5 Å to 2.4 Å including improvements in thecrystals. A back-extraction procedure was developed for reproduciblygrowing crystals that are much thicker than those available earlier andcan diffract to 2.2 Å resolution (see Example 1). Briefly, the crystalswere grown at room temperature in hanging drops by vapor diffusion fromseeded solutions back-extracted from precipitated subunits. The crystalsthat resulted had maximum dimensions of 0.5×0.5×0.2 mm and wereharvested after three weeks. The twinning of crystals that obstructedprogress for many years (Ban et al. (1999) supra) was eliminated byadjusting crystal stabilization conditions (see Example 1). Crystalswere stabilized by gradual transfer into a solution containing 12%, PEG6000, 22% ethylene glycol, 1.7 M NaCl, 0.5 M NH₄Cl, 100 mM potassiumacetate, 30 mM MgCl₂ and 1 mM CdCl₂, pH 6.2, and flash frozen in liquidpropane. Reducing the salt concentration below 1.7 M NaCl (KCl)increased the tendency of crystals to become twinned. At saltconcentrations as low as 1.2 M nearly all of the crystals were twinned.

[0149] All the X-ray data used for high resolution phasing werecollected at the Brookhaven National Synchrotron Light Source except fortwo native data sets used, which were collected at the Advanced PhotonSource at Argonne (see Example 2) (Table 1). Osmium pentamine (132sites) and Iridium hexamine (84 sites) derivatives proved to be the mosteffective in producing both isomorphous replacement and anomalousscattering phase information to 3.2 Å resolution (see Example 2).Inter-crystal density averaging which had contributed significantly atlower resolution, was not helpful beyond about 5 Å resolution. Electrondensity maps were dramatically improved and their resolutions extended,eventually to 2.4 Å, using the solvent flipping procedure in CNS(Abrahams et al. (1996) Acta Crystollogr. D 52: 30; Brünger et al.(1998) Acta Crystallogr. D Biol. Crystallogr. 54: 905-921). TABLE 1Statistics for Data Collection, Phase Determination, and ModelConstruction Data Statistics MIRAS1 MIRAS2 Native1 Os(NH₃)₅ ²⁺ UO₂F₅ ³⁻Native2 Ir(NH₃)₆ ³⁺ Os(NH₃)₆ ³⁺ Ta₆Br₁₂ ²⁺ Heavy atom —      30.0     05 —      20 0      4 5      3.0 conc. (mM) Soaking time (hrs) —      1 5    4 —   24 hrs   24 hrs   24 hrs Sites no —     132    20 —      84     38      9 Resolution (Å) 90-24 40-3 5 40-3 8 30-2 9 30-3 2 30-3.530-3 8 (*) (2.5-2.4) (3 6-3 5) (3.9-3.8) (3.0-2 9) (3 32-3 22) (3.27-320) (3 6-3 5) (3 97-3 80) λ(Å)      1 00      1 14     1.30      1.00     1.075      1 14      1 255 Observations 6,089,802 1,308,703 596,1662,832,360 1,823,861 1,646,468 1,288,524 Unique   665,928   429,761313,863   390,770   541,488   488,275   346,745 Redun. (*)  9 1 (6.5)  30 (2 5)  1.9 (1.6)      7 2      3.4  4 3 (4 2)      3 7 Completeness(*) 95 6 (71 0) 99 4 (96 8) 92.0 (54 1)      97 1      93 8 98 1 (99 0)     99 5 I/σI (Last bin) 25 5 (1 9) 13 5 (3 3)  8 9 (1 6) 18 0 (6 4) 120 (2.6) 10.6 (2 7) 10 8 (3 2) R_(mcrgc) (*)  8.6 (69 1)  7 2 (32 0)  9 1(37 9) 11 2 (36 9)  8 5 (29 5) 12.1 (46.0) 12.1 (40.5) 02 (ano) (*) — 2.8 (1 0)  1 5 (1 0) — 2.63 (1.48)  1.8 (1 0) 2 42 (1.18) R_(mergc)(ano) —      6 2     8 0 —      6 7      6 9 R_(iso) — 14 1 (22 7) 26 4(47 0) — 12 9 (28 1) 19 5 (39 4) Phasing Statistics Resolution shells(Å): ˜73,200 reflections per bin 30.0 5.1 4.0 3.5 3.2 Total MIRAS1 (FOM)0 52 0 31 0 14 — 0 32 Os(NH₃)₅ ²⁺ Phasing power 0 87 0.72 0 66 — 0 75Phasing power (SAD) 1 40 0 58 0 26 — 0 75 R_(cullis) (centric) 0 62 0 650 67 — 0 65 UO₂F₅ ³⁻ Phasing Power 0 47 0 33 0 28 — 0 36 Phasing power(SAD) 0 46 0 25 — — 0 36 R_(cullis) (centric) 0 72 0 77 0 75 — 0 75MIRAS2 (FOM) 0 48 0 40 0 28 0 12 0 33 Ir(NH₃)₆ ³⁺ Phasing power 1 02 092 0 78 0 66 0 89 Phasing power (SAD) 2 02 1 60 1.22 0 83 1 47R_(cullis) (centric) 0 58 0 63 0 70 0 74 0 63 Os(NH₃)₆ ³⁺ Phasing power0 62 0 57 0 58 0 58 0 59 Phasing power (SAD) 0 47 0 39 — — 0 42R_(cullis) (centric) 0 78 0 78 0 78 0 76 0 78 Ta₆Br₁₂ ²⁺ (Used for SADphasing only) Phasing Power (SAD) 2 77 0 35 0 13 — 1 19FOM_((MIRAS1 + MIRAS2 + SAD)) 0.76 0 51 0 31 0 14 0 37 Model Statisticsrms deviations Average B factors (Å²) Resolution range (Å) 90 0-24 Bonds(Å)  0.0064 All atoms 37 4 Reflections 577,304 Angles (°)  1 19 23S rRNA32 3 R_(sryst) (%)    25 2 Dihedrals (°) 28 8 5S rRNA 43 2 R_(free) (%)   26 1 Impropers (°)  1 68 Minimum/Max B factors (Å²) 70/107 9

[0150] Except for regions obscured by disorder, theexperimentally-phased, 2.4 Å resolution electron density map was ofsufficient quality so that both protein and nucleic acid sequencingerrors could be identified and corrected. Each nucleotide could befitted individually and the difference between A and G was usually clearwithout reference to the chemical sequence, as was the distinctionbetween purines and pyrimidines (FIG. 1).

[0151] Subtraction of the atomic model from the experimental electrondensity map leaves no significant density except for water and ions,showing that the model accounts for all the macromolecular density.Preliminary refinement of the model was achieved using a mixed target inthe program CNS (Brünger et al. (1998) supra). The model was furtherrefined in real space against the 2.4 Å electron density map using theprogram TNT (Tronrud (1997), Macromolecular Crystallogaphy, Part B,Methods In Enzymology), which yielded a model with a free R-factor of0.33. One additional round of mixed target refinement of both atomicpositions and B-factors using CNS led to the structure described below.Its free R-factor is 0.27 (Table 1).

[0152] 2. Sequence Fitting and Protein Identification.

[0153] The sequence of 23S rRNA was fit into the electron density mapnucleotide by nucleotide starting from its sarcin/ricin loop sequence(A2691-A2702) (E. coli numbers A2654 to A2665), whose position had beendetermined at 5 Å resolution (Ban et al. (1999) supra). Guided by theinformation available about the secondary structures of 23S rRNAs(Gutell, R. R. (1996), “Comparative Sequence Analysis and the Structureof 16S and 23S rRNA,” Ribosomal RNA. Structure, Evolution, Processing,and Function in Protein Biosynthesis, (Dahlberg A. and Zimmerman B.,eds.), CRC Press, Boca Raton, Fla. pp. 111-128 ), the remaining RNAelectron density neatly accommodated the sequence of 5S rRNA. Theinterpretation of protein electron density corresponding to the proteinwas more complicated because each protein region had to be identifiedchemically before the appropriate sequence could be fit into it, butabout 4,000 amino acid residues were fit into electron density.

[0154] The H. marismortui 50S subunit appears to contain thirty-oneproteins, and there are sequences in the Swiss-Prot data bank for twentyeight of those thirty one proteins, including one, HMS6 or L7ae, thatwas originally assigned to the small ribosomal subunit(Whittmann-Liebold et al. (1990) supra). The three remaining proteinswere identified using the sequences of the ribosomal proteins fromeukaryotes and other archeal species as guides. No electron density wasfound for one of the H. marismortui large ribosomal subunit proteins inthe sequence database, LX. Either the assignment of LX to the largesubunit is in error, or LX is associated with a disordered region of thesubunit, or LX is absent from the subunits examined altogether.

[0155] The 2.4 Å resolution electron density map lacks clear electrondensity for proteins L1, L10, L11 and L12, the positions of which areknown from earlier low resolution X-ray and/or electron microscopicstudies. These proteins are components of the two lateral protuberancesof the subunit, which are both poorly ordered in these crystals. L1 isthe sole protein component of one of them (Oakes, M. et al. (1986)),Structure, Function and Genetics of Ribosomes, (Hardesty, B. and Kramer,G., eds.) Springer-Verlag, New York, N.Y., 47-67) and is evident in 9 Åresolution density maps of the subunit (Ban et al. (1998) supra), butnot at higher resolutions. L10, L11 and L12 are components of the otherprotuberance, which is often referred to as the L7/L12 “stalk” (Oakes etal. (1986) supra). L11 and the RNA to which it binds were located in the5 Å resolution electron density map of the H. marismortui large subunit(Ban et al. (1999) supra) using the independently determined crystalstructures of that complex (Conn G L et al. (1999) Science284:1171-1174; Wimberly et al. (1999) Cell 97: 491-502). A proteinfragment (about 100 residues) that is associated with the RNA stalk thatsupports the L11 complex can be seen in the 2.4 Å resolution map. Basedon location, it must be part of L10. There is no electron densitycorresponding to L12 seen at any resolution, but the L12 tetramer isknown to be attached to the ribosome through L10, and the L10/L12assembly is known to be flexible under some circumstances (Moller et al.(1986) Structure, Function, and Genetics of Ribosomes, supra,pp.309-325), which may explain its invisibility here.

[0156] The structures of eubacterial homologues of proteins L2, L4, L6,L14, and L22 have previously been determined in whole or in part (see,Table 2). L2, L6 and L14 were initially located in the 5 Å resolutionmap (Ban et al. (1999) supra). L4 and L22 have now been identified andpositioned the same way. Electron density corresponding to most of theremaining proteins was assigned by comparing chain lengths and sequencemotifs deduced from the electron density map with known sequencelengths, guided by the information available about relative proteinpositions (Walleczek et al. (1988) EMBO J. 7: 3571-3576) and proteininteractions with 23S rRNA and 5S rRNA (Ostergaard et al. (1998) J. Mol.Biol. 284: 227-240). Each of the protein electron density regions soidentified is well accounted for by its amino acid sequence.

[0157] The most interesting of the proteins identified by sequencesimilarity was L7ae, which first appeared to be L30e. The L30eidentification seemed plausible because the structure of yeast L30superimposes neatly on the electron density of L7ae, and the structureof the RNA to which L7ae binds closely resembles that of the RNA towhich yeast L30 binds (Mao, H. et al. (1999) Nat. Struct. Biol. 6:1139-1147). Nevertheless, the sequence of HMS6, which by sequencesimilarity is a member of the L7ae protein family, fits the electrondensity better. Four of the other proteins identified by sequencesimilarity, L24e, L37e, L37ae, and L44e, contain zinc finger motifs. Therat homologues of L37e and L37ae were predicted to be zinc fingerproteins on the basis of their sequences (Wool et al. (1995) supra), andthis prediction helped identify their homologues in H. marismortui.TABLE 2 Large Subunit Proteins from Haloarcula Marismortui Interactions⁵Name¹ Hmlg² Lgth³ Conf⁴ 1 2 3 4 5 6 5S Proteins L1* 211 glb. + none L2†RL8 239 glb + ext + + + + (L37ae) L3 RL3 337 glb + ext + + + + L14,L24e, (L13) L4† RL4 246 glb + ext + + Å (L18e), (L24), (L37e) L5 RL11176 glb + + L18 L6 RL9 177 glb Å Å + (L13) L10* RP0 348 glb? + L12 L11*RL12 161 glb + none L12* RL1/2 115 glb L10 L13 RL13a 145 glb + Å Å (L3),(L6) L14 RL23 132 glb + + + L3, L24e L15 RL27a 164 glb + ext + + +(L18e), (L32e) L18 RL5 186 glb + ext Å + + L5, L21e L19 RL19 148 glb +ext + + + Å none L22 RL17 154 glb + ext + Å + + + + none L23 RL23a 84glb Å + L29, (L39e) L24 RL26 119 glb + ext + (L4) L29 RL35 70 glb + L23L30 RL7 154 glb + + none L18e RL18 115 glb + (L4), (L15) L21e RL21 95glb + + Å L18 L24e RL24 66 glb Å + L3, L14 L31e RL31 91 glb + + + noneL32e RL32 240 glb Å + (L15) L37e RL37 56 glb + ext + + + Å (L4) L39eRL39 49 ext + + (L23) L44e RL36a 92 glb + ext + Å + (L15e) L7ae R17a 110glb Å L15e L10e RL10 163 glb + + Å none L15e RL15 184 glb + ext + Å ÅÅ + (L44e), L7ae L37ae RL37a 72 glb + ext + + + L2 # (1980) Nature 286:824-827), L14 (Davies et al. (1996) Structure 4: 55-66), L22 (Unge etal. (1998) Structure 6: 1577-1586), L30 (Wilson et al. (1986) Proc. Nat.Acad. Sci. USA 83: 7251-7255). All other structures, except 10, havebeen newly determined in this study.

[0158] 3. General Appearance of the Subunit.

[0159] In its crown view (see FIG. 2), the large ribosomal subunit,which is about 250 Å across, presents its surface that interacts withthe small subunit to the viewer with the three projections that radiatefrom that surface pointed up. Although the protuberance that includes L1is not visible in the 2.4 Å resolution electron density map, thestructure of L1, which has been determined independently (Nikonov et al.(1996) EMBO J. 15: 1350-1359), has been positioned approximately inlower resolution maps (Ban et al. (1998) supra) and is included here toorient the reader. It is evident that, except for its two lateralprotuberances, the large ribosomal subunit is monolithic. There is nohint of a division of its structure into topologically separate domains.In addition, partly because it lacks obvious domain substructure butalso because it is so large, it is impossible to comprehend looking atit as a whole. In order to convey to the reader a sense of how it is puttogether, the subunit must be dissected into its chemical components.

[0160] 4. RNA Secondary Structure.

[0161] All the base pairs in H. marismortui 23S rRNA stabilized by atleast two hydrogen bonds were identified using a computer program thatsearched the structure for hydrogen bond donors and acceptors separatedby less than 3.2 Å. Bases linked by at least two such bonds wereconsidered paired if the angle between their normals was less than 45°,and the angle between bonds and base normals was also less than 45°.Based on the results of this analysis, a secondary structure diagram hasbeen prepared in the format standard for 23S/28S rRNAs (see FIG. 3). Thesecondary structure predicted for this molecule by phylogeneticcomparison was remarkably accurate, but it did not find all of thetertiary pairings and failed to identify interactions involvingconserved bases. In addition to base pairs of nearly every type, the RNAcontains numerous examples of well-known secondary structure motifs suchas base triplets, tetraloops, and cross-strand purine stacks, but nodramatically new secondary structure motifs have been identified so far.

[0162] The secondary structure of this 23S rRNA consists of a centralloop that is closed by a terminal stem, from which 11 more or lesscomplicated stem/loops radiate. It is customary to describe the moleculeas consisting of 6 domains, and to number its helical stems sequentiallystarting from the 5′ end (see FIG. 4) (Leffers et al. (1987) supra). Thedivision of the molecule into domains shown in FIG. 4 deviates fromstandard practice with respect to helix 25, which is usually consideredto be part of domain I. It is placed in domain II because it interactsmore strongly with domain II than it does with the other elements ofdomain I.

[0163] There are five sequences longer than 10 nucleotides in 23S rRNAwhose structures cannot be determined from the 2.4 Å resolution map dueto disorder. Altogether they account for 207 out of the 232 nucleotidesmissing from the final model. The disordered regions are: (1) all ofhelix 1, (2) the distal end of helix 38, (3) helix 43/44 to whichribosomal protein L11 binds, (4) the loop end of stem/loop 69, and (5)helix 76/77/78, which is the RNA structure to which L1 binds. Forcompleteness, these regions are included in FIG. 3 (in gray) with theirsecondary structures determined phylogenetically.

[0164] 5. Overall Architecture of rRNA.

[0165] The six domains of 23S rRNA and 5S rRNA all have complicated,convoluted shapes that nevertheless fit together to produce a compact,monolithic RNA mass (see FIGS. 4(A) and 4(B)). Thus despite theorganization of its RNAs at the secondary structure level, inthree-dimensions, the large subunit is a single, gigantic domain. Inthis respect, it is quite different from the small subunit, which is aflatter object that is not at all monolithic. Even in low resolutionelectron micrographs the small subunit consists of three structuraldomains, each of which, it turns out, contains one of the threesecondary structure domain of its RNA (Noller et al. (1990) TheRibosome: Structure, Function, and Evolution, supra, pp. 73-92). Thisqualitative difference between the two subunits may reflect arequirement for conformational flexibility that is greater for the smallsubunit.

[0166] Domain I, which looks like a mushroom (see FIG. 4(E)), lies inthe back of the particle, behind and below the L1 region. The thin partof the domain starts in the vicinity of domain VI, which is where itsfirst and last residues are located. Helices 1 and 25 span the particlein the back and then the domain expands into a larger, more globularstructure below and behind the L1 region.

[0167] The largest of the six 23S rRNA domains, domain II, whichaccounts for most of the back of the particle, has three protrusionsthat reach towards the subunit interface side of the particle (see FIG.4(F)). One of them (helix 42-44) is the RNA portion of the L7/L12 stalk,which is known to interact with elongation factors, is not well-orderedin these crystals. The second domain II protrusion is helix 38, which isthe longest, unbranched stem in the particle. It starts in the back ofthe particle, bends by about 90 degrees and protrudes towards the smallsubunit between domains V and 5S rRNA. The third region, helix 32-35.1,points directly towards the small subunit and its terminus, the loop ofstem/loop 34, interacts directly with the small ribosomal subunit(Culver et al. (1999) Science 285: 2133-2135). This loop emerges at thesubunit interface between domains III and IV.

[0168] Domain III is a compact globular domain that occupies the bottomleft region of the subunit in the crown view (see FIG. 4(G)). It lookslike a four pointed star with the origin of the domain (stem/loop 48)and stem/loops 52, 57, and 58 forming the points. The most extensivecontacts of domain III are with domain II, but it also interacts withdomains I, IV and VI. Unlike all the other domains, domain III hardlyinteracts with domain V at all; the sole contact is a single van derWaals contact involving a single base from each domain.

[0169] Domain IV accounts for most of the interface surface of the 50Ssubunit that contacts the 30S subunit (see FIG. 4(H)). It forms a largediagonal patch of flat surface on that side of the subunit, and connectsto domains III and V in the back of the particle. Helices 67-71 are themost prominent feature of domain IV, and form the front rim of theactive site cleft, which is clearly visible at low resolution (see FIG.2). This is one of the few regions of the 23S rRNA that is notextensively stabilized by ribosomal proteins. Helix 69 in the middle ofthis ridge interacts with the long penultimate stem of 16S rRNA in thesmall ribosomal subunit and can be viewed as a divider separating A-sitebound tRNAs from P-site bound tRNAs.

[0170] Domain V, which is sandwiched between domains IV and II in themiddle of the subunit, is known to be intimately involved in thepeptidyl transferase activity of the ribosome. Structurally the domaincan be divided into three regions (see FIGS. 4(I) and 4(J)). The firststarts with helix 75 and ultimately forms the binding site for proteinL1. The second, which consists of helices 80-88, forms the bulk of thecentral protuberance region, and is supported in the back by the 5S rRNAand domain II. The third region, which includes helices 89-93, extendstowards domain VI and helps stabilize the elongation factor bindingregion of the ribosome.

[0171] The smallest domain in 23S rRNA, domain VI, which forms a largepart of the surface of the subunit immediately below the L7/L12 stalk,resembles a letter X with a horizontal bar at the bottom (see FIG.4(K)). An interesting region of this domain is the sarcin-ricin loop(SRL) (stem/loop 95), the structure of which has been extensivelystudied in isolation (Szewczak et al. (1995) J. Mol. Biol. 247: 81-98).The SRL is essential for factor binding, and ribosomes can beinactivated by the cleavage of single covalent bonds in this loop (Woolet al. (1992) TIBS 17: 266-269). As suggested by nucleotide protectiondata, the major groove of this loop is exposed to solvent (Moazed et al.(1988) Nature 334: 362-364), and its conformation is stabilized byproteins and through interaction with domain V that involves two baseson the minor grove side. The nucleotides involved are A 2699 and G 2700in domain VI, and A 2566 and G 2567 in domain V.

[0172] 5S ribosomal RNA, which is effectively the seventh RNA domain inthe subunit, consists of three stems radiating out from a commonjunction called loop A (see FIG. 4(D)). In contrast to what is seen inthe crystal structure of fragment 1 from E. coli 5S rRNA (Correll et al.(1997) Cell 91: 705-712), the helix 2/3 arm of the molecule stacks onits helix 4/5 arm, not helix 1 (see FIG. 4(L)). This arrangement resultsfrom a contorted conformation of loop A residues that involves twostacked base triples. Indeed, from the secondary structure point ofview, the loopA-helix 2,3 arm of 5S rRNA is quite remarkable. Nowhereelse in the subunit is there a higher concentration of unusual pairingsand of convoluted RNA secondary structure.

[0173] 6. Sequence Conservation and Interactions in 235 rRNA.

[0174] While 23S/28S rRNAs contain many conserved sequences, they alsovary substantially in chain length. Shorter 23S/28S rRNAs aredistinguished from their longer homologues by the truncation of, or eventhe elimination of entire stem/loops, and by comparing sequences, onecan identify a minimal structure that is shared by all (Gerbi (1995)Ribosomal RNA: Structure, Evolution, Processing and Function in ProteinBiosynthesis, supra, pp. 77-88). The expansion sequences in the 23S rRNAof H. marismortui, i.e., the sequences it contains that are larger thanthe minimum, are shown in FIG. 5 in green. They are largely absent fromthe subunit interface surface of the particle, but they are abundant onits back surface, far from its active sites. This is consistent with lowresolution electron microscopic observations suggesting that the regionof the large subunit whose structure is most conserved is the surfacethat interacts with the small subunit (Dube et al. (1998) Structure 6:389-399).

[0175] There are two classes of conserved sequences in 23S rRNA. Onecontains residues concentrated in the active site regions of the largesubunit. The second class consists of much shorter sequences scatteredthroughout the particle (FIG. 5: red sequences). The SRL sequence indomain VI and the cluster of conserved residues belonging to domain Vthat are located at the bottom of the peptidyl transferase cleft aremembers of the first class. They are conserved because they areessential for substrate binding, factor binding and catalytic activity.Most of the residues in the second class of conserved residues areinvolved in the inter- and intra-domain interactions that stabilize thetertiary structure of 23S rRNA. Adenosines are disproportionatelyrepresented in this class. The predominance of A's among the conservedresidues in rRNAs has been pointed out in the past (Ware et al. (1983)Nucl. Acids. Res. 22: 7795-7817).

[0176] In addition to its reliance on A-dependent motifs, the tertiarystructure of the domains of 23S rRNA and their relative positions arestabilized by familiar tertiary structure elements like RNA zippers andtetraloop/tetraloop receptor motifs (Moore, P. B. (1999) Annu. Rev.Biochem. 68: 287-300), and in many places, base pairs and triplesstabilize the interactions of sequences belonging to differentcomponents of the secondary structure of 23S rRNA.

[0177] Interestingly, 5S rRNA and 23S rRNA do not interact extensivelywith each other. The few RNA/RNA interactions there are involve thebackbones of the helix 4/5 arm of 5S rRNA and the backbone of helix 38of 23S rRNA. Most of the free energy and all of the specificity of 5SrRNA binding to the large ribosomal subunit appears to depend on itsextensive interactions with proteins that act as modeling clay stickingit to the rest of ribosome.

[0178] 7. Proteins in the 50S Ribosomal Subunit.

[0179] The structures of twenty seven proteins found in the largeribosomal subunit of H. marismortui (Table 2) have been determined.Twenty-one of these protein structures have not been previouslyestablished for any homologues, and the structures of the six that dohave homologues of known structure have been rebuilt into the electrondensity map using their H. marismortui sequences. In addition, there arestructures available for homologues of H. marismortui L1, L11 and L12,which cannot be visualized in the 2.4 Å resolution electron density map.Only the structure of L10 is still unknown among the thirty one proteinsknown to be present.

[0180] Not every one of these structures is complete. For example, anentire domain of L5 is missing from the electron density, presumablybecause of disorder. L32e is also noteworthy in this regard. Abouttwenty residues from its N-terminus are not seen in the electron densitymap, and the electron density map suggests that its C-terminal residueis covalently bound to the most N-terminal of its visible residues.

[0181] Of the thirty large subunit ribosomal proteins whose structuresare known, 17 are globular proteins, similar in character to thousandswhose structures are in the Protein Data Bank (Table 2). The remainingthirteen proteins either have globular bodies with extensions protrudingfrom them (“glb+ext”) or are entirely extended (“ext”). Their extensionsoften lack obvious tertiary structure and in many regions are devoid ofsecondary structure as well (see FIG. 6). These extensions may explainwhy many ribosomal proteins have resisted crystallization in isolation.The exceptions that prove the rule are L2 and L4, both of which areproteins belonging to the “glb+ext” class. Protein L2 was crystallizedand its structure solved only after its extensions had been removed(Nakagawa et al. (1999) supra), and one of the two regions of L4 thatare extended in the ribosome is disordered in the crystal structure ofintact L4 (Wahl et al. (2000) supra).

[0182] Except for proteins L1, L7, L10 and L11, which form the tips ofthe two lateral protuberances, the proteins of the 50S subunit do notextend significantly beyond the envelope defined by the RNA (see FIG.7). Their globular domains are found largely on the particle's exterior,often nestled in the gaps and crevices formed by the folding of the RNA.Thus, unlike the proteins in spherical viruses, the proteins of thelarge ribosomal subunit do not form a shell around the nucleic acid withwhich they associate, and unlike the proteins in nucleosomes, they donot become surrounded by nucleic acid either. Instead, the proteins actlike mortar filling the gaps and cracks between “RNA bricks”.

[0183] The distribution of proteins on the subunit surface is nearlyuniform, except for the active site cleft and the flat surface thatinteracts with the 30S subunit. In the crown view the proteins liearound at the periphery of the subunit (see FIG. 7(A)), but when viewedfrom the side opposite the 30S subunit binding site (the “back side”),they appear to form an almost uniform lattice over its entire surface(see FIG. 7(B)). Similarly, the bottom surface of the subunit, whichincludes the exit of polypeptide tunnel, is studded with proteins (seeFIG. 7(C)). Indeed, the 5 proteins that surround the tunnel exit mayplay a role in protein secretion since they are part of the surface thatfaces the membrane and the translocon when membrane and secretedproteins are being synthesized.

[0184] Although FIG. 7 shows protein chains disappearing into theribosome interior, the degree to which proteins penetrate the body ofthe particle can only be fully appreciated when the RNA is strippedaway. The interior of the particle is not protein-free, but it isprotein-poor compared to the surface of the particle. Extended tentaclesof polypeptide, many of which emanate from globular domains on thesurface, penetrate into the interior, filling the gaps betweenneighboring elements of RNA secondary structure (see FIG. 8(E)). Thebizarre structures of these extensions are explained by theirinteractions with RNA.

[0185] Although extended, non-globular structures are rare in theprotein data base, they are not unknown. Extended protein termini oftenform inter-protein contacts, e.g., in viral capsids, presumably adoptingfixed structures only upon capsid formation. The basic “tails” ofhistones may behave the same way when nucleosomes form. The N-terminalsequences of capsid proteins are often positively charged, and in viruscrystal structures, the electron density for these sequences oftendisappears into the interior of the virus where these sequencespresumably interact with asymmetrically arranged nucleic acid. Theinteractions observed in the ribosome could be useful models for theseviral interactions.

[0186] The interactions of extended polypeptides and RNA in the largesubunit, which stabilizes its massive nucleic acid structure, result ina tangle of RNA and protein in the center of the subunit (see FIGS. 8(A)and 8(B)). It is hard to imagine such an object assembling from itscomponents efficiently in anything other than a highly ordered manner.Chaperones may well be required to prevent the aggregation of theextended regions of these proteins, which are likely to be disorderedoutside the context provided by rRNA, and to manage the folding of rRNA.

[0187] 8. Protein and RNA Interactions.

[0188] Because protein permeates the large subunit to a surprisingdegree, there are only a few segments of the 23S rRNA that do notinteract with protein at all. Of the 2923 nucleotides in 23S rRNA, 1157nucleotides make at least van der Waals contact with protein (see FIG.8(D)), and there are only ten sequences longer than twenty nucleotidesin which no nucleotide contacts protein. The longest such sequencecontains forty-seven nucleotides, and is the part of domain IV thatforms the ridge of the active site cleft.

[0189] The extent of the interactions between RNA and protein that occurwhen the large subunit assembles can estimated quantitatively. Using theRichards algorithm (Lee, B. et al. (1971) J. Mol. Biol. 55: 379-400) anda 1.7 Å radius probe to compute accessible surface areas, it can beshown that 180,000 Å² of surface becomes buried when the subunit formsfrom its isolated, but fully structured components. This is about halftheir total surface area. The average is about 6,000 Å² per protein.While this is an enormous amount compared to the surface buried whenmost protein oligomers form, it should be recognized that ribosomeassembly must be accompanied by a large loss in conformational entropythat does not occur when most proteins oligomerize. The extended proteintermini and loops of the ribosomal proteins are almost certainlyflexible in isolation, and in the absence of protein, the RNA isprobably quite flexible as well. Thus, the burial of a large amount ofsurface area may be required to provide the energy necessary to fix ofthe structures of these molecules.

[0190] All of the proteins in the particle except L12, interact directlywith RNA and all but seven of the remaining thirty proteins interactwith two rRNA domains or more (Table 2). The “champion” in this regardis L22, which is the only protein that interacts with RNA sequencesbelonging to all 6 domains of the 23S rRNA (see FIG. 8(C)). Theprotein-mediated interactions between 5S rRNA and 23S rRNA areparticularly extensive. Protein L18 attaches helix 1 and helix 2/3 of 5SrRNA to helix 87 of 23S rRNA. Protein L3le mediates an interactionbetween the same part of 5S rRNA and domains II and V. Loop C is linkedto domain V by protein L5 and loop D is attached to domains II and V byprotein L10e. Whatever else they may do, it is evident that an importantfunction of these proteins is stabilization of the relative orientationsof adjacent RNA domains. Several also help secure the tertiarystructures of the domains with which they interact.

[0191] Since most ribosomal proteins interact with many RNA sequencesand the number of proteins greatly exceeds the number of RNA domains, itcan hardly come as a surprise that every rRNA domain interacts withmultiple proteins (Table 2). Domain V, for example, interacts withthirteen proteins, some intimately and a few in passing.

[0192] It is clear that the oligonucleotide binding experiments longrelied on for information about the RNA binding properties of ribosomalproteins have underestimated their potential for interacting with RNA.The high-affinity RNA binding site identified on a protein by such anexperiment may indeed be important for ribosome assembly, but its many,weaker interactions with other sequences are likely to be missed, andthey too may be vital for ribosome structure. Most ribosomal proteinscrosslink RNA and crosslinking is impossible without multipleinteractions. Similar considerations may apply to proteins that arecomponents of other ribonucleoproteins such as the sliceosome.

[0193] Of the seven proteins that interact with only one domain, three(L1, L10, L11) participate directly in the protein synthesis process.Rather than being included in the ribosome to ensure that the RNA adoptsthe proper conformation, it seems more appropriate to view the RNA asbeing structured to ensure their correct placement. Another three (L24,L29, L18e) interact with several secondary structure elements within thedomains to which they bind, and presumably function to stabilize thetertiary structures of their domains. The last of the single RNA domainproteins, L7ae, is puzzling. On the one hand, it cannot function as anRNA stabilizing protein because it interacts with only a single, shortsequence in domain I, and on the other hand, it is far from theimportant functional sites in the subunit, the peptidyl transferase siteand factor binding site. It is quite close to L1, however, which appearsto be important for E-site function (Agrawal et al. (1999) J. Biol.Chem. 274: 8723-8729), and maybe it is involved in that activity.

[0194] While many ribosomal proteins interact primarily with RNA, a fewinteract significantly with other proteins. The most striking structuregenerated by protein-protein interactions is the protein clustercomposed of L3, L6, L14, L19 and L24e that is found close to the factorbinding site. The protein surface they provide may be important forfactor interactions.

[0195] The structure presented above illuminates both the strengths andweaknesses of approaches to complex assemblies that depend ondetermining the structures of components in isolation. The structures ofthe globular domains of homologues of the proteins in large ribosomalsubunit from H. marismortui are largely the same as those of thecorresponding domains in the intact subunit, though adjustments indomain positions are sometimes required. Consequently, these structureswere very useful for locating proteins and interpreting lower resolutionelectron density maps. However, for obvious reasons, the structures ofthe extended tails and loops of ribosomal proteins cannot be determinedin the absence of the RNAs that give them structure, and the feasibilityof strategies that depend on producing low molecular weight RNA-proteincomplexes that have all the RNA contacts required to fix the structuresof such proteins seems remote. RNA is also a problem. While thesarcin/ricin loop has much the same structure in isolation as it does inthe ribosome, the structure of 5S rRNA in isolation differs in somerespects from what is seen in the ribosome, and the structure of theisolated P-loop (Puglisi et al. (1997) Nat. Struct. Biol. 4: 775-778)does not resemble the structure of the P-loop in the ribosome at all.Clearly a “structural genomics” approach to the ribosome, which wouldhave entailed determining the structures of all its proteins and allpossible rRNA fragments, would not have worked. It may not be successfulfor other macromolecular assemblies either.

[0196] B. The Structural Basis of Ribosome Activity in Peptide BondSynthesis

[0197] Analysis of the atomic co-ordinates discussed in section IIAabove together with additional atomic co-ordinates of a ribosomalsubunit complexed with various analogues, similarly refined, permit ananalysis of ribosome function. Accordingly, the present invention isalso based on the crystals of Haloarcula marismortui 50S ribosomalsubunit complexed either with the Yarus transition state analogue,CCdA-p-Puro, or with a mini-helix analogue of an aminoacyl-tRNA. Thepresent invention provides the structures of both complexes. The atomicco-ordinates of the structure of both complexes were deposited on Jul.26, 2000, at Research Collaboratory for Structural Bioinformatics (RCSB)Protein Data Bank (PDB) (Berman et al. (2000) Nucleic Acid Research 28:235-242; http://www.rcsb.org/pdb/) with accession numbers PDB ID: 1FFZ(50 ribosome/CCdA-p-Puro complex) and PDB ID: 1FGO (50 ribosome/aa-tRNAanalogue).

[0198] As discussed below, the complete atomic structures of the largeribosomal subunit and its complexes with two substrate analogues showthat the ribosome is a ribozyme. The complete atomic structures alsoprovide information regarding the catalytic properties of its all-RNAactive site. Both substrate analogues are contacted exclusively byconserved rRNA residues from domain V of 23S rRNA; there are no proteinside-chains closer than about 18 Å to the peptide bond beingsynthesized. The mechanism of peptide bond synthesis appears to resemblethe reverse of the deacylation step in serine proteases, with the baseof A2486 (A2451) in E. coli playing the same general base role as His57in chymotrypsin. The unusual pKa that A2486 must possess to perform thisfunction probably derives from its hydrogen bonding to G2482 (G2447)which interacts with a buried phosphate that could stabilize the unusualtautomers of two bases which is required for catalysis. The polypeptideexit tunnel is largely formed by RNA but has significant contributionsfrom proteins L22, L39 and L4 and its exit is encircled by proteins L19,L22, L23a, L24 and L29.

[0199] The CCdA from the Yarus analogue binds to the so-called P-loopand hence must be in the P-site. Only the terminal-CCA of the aa-tRNAanalogue is visible, but since it interacts appropriately with theA-loop (Kim et al (1999) Molec. Cell 4: 859-864), it must be in theA-site. The puromycin group occupies the same location in bothstructures, and there are no proteins near that site. Hence, thecatalytic activity of the active site must depend entirely on RNA. TheN3 of A2486 (E. coli A2451) is the titratable group nearest to thepeptide bond being synthesized and is likely functioning as a generalbase to facilitate the nucleophilic attack by the α-amino group of theA-site substrate. In order to function in this capacity, the pKa of thisbase has to be roughly 5 units higher than normal.

[0200] 1. Structures of Substrate Analogue Complexes.

[0201] In order to establish how substrates interact at the A-site andP-site of the large subunit, two substrate analogues were used. One ofthe analogues, which was designed to mimic the acceptor stem of anaa-tRNA and bind to the A-site, was a twelve base-pair RNA hairpin withan aminoacylated, four-nucleotide extension on its 3′ end (see FIG. 9).The sequence used was that of the tRNA tyr acceptor stem and it isterminated with puromycin, which itself is an analogue of tyrosyl-A76.The second analogue used was the Yarus transition state analogue,CCdA-p-puromycin. As in the case of the A-site substrate analogue, thepuromycin of the Yarus inhibitor is expected to bind at the A-site,while its CCdA moiety should bind at the P-site.

[0202] The positions of the Yarus inhibitor and the tRNA acceptor stemanalogue were determined by soaking these molecules into crystals of theH. marismortui 50S ribosomal subunit, measuring diffraction data to 3.2Å resolution and calculating difference electron density maps (Welch etal. (1997) Biochemistry 36: 6614-6623). Maps of the complexes were alsocalculated using 2F_(o)(complexed)-F_(o)(uncomplexed) as coefficients,to examine the shifts in the positions of ribosome residues that occurwhen these analogues bind (see FIG. 10(B) and Table 3). TABLE 3Statistics for Data Collection and Scaling. Crystal Native A Native BCcdAp-Puro Mini-helix Soak time (hours) — — 24 24 Soak concentration(μM) — — 100 100 Wavelength (Å) 1.0 1.0 1.0 1.0 Observations 1,571,1711,344,877 2,590,726 2,712,813 Unique 284,533 369,167 367,284 447,121Redundancy 5.5 3.6 7.0 6.0 Resolution limits (Å) 70.0 − 3.2 70.0 − 3.070.0 − 3.0 70.0 − 2.8 (High-resolution bin)* (3.26 − 3.20) (3.05 − 3.00)(3.23 − 3.17) (3.08 − 3.02) Completeness 94.1 (96.0) 98.9 (99.3) 98.6(99.9) 99.6 (100) 1/σl 14.6(4.0) 10.8(3.1) 11.0(2.8) 10.7(2.9)R_(merge)† 10.2 (40) 11.5 (38) 18.8 (84) 14.3 (72) R_(iso) Native A‡ — —6.8 (20.8) 14.4 (25.2) R_(iso) Native B‡ — — 12.6(27.4) 17.5(31.0)†R_(merge): ΣΣ_(i)/I_((h)) − I_((h)l)|IΣΣI_((h)i)′ where I_((h)) is themean intensity after reflection.

[0203] A model for the entire Yarus inhibitor could be fitted into thedifference density (see FIG. 10(A)), and the electron density map of thecomplex shows the N3 of A2486 (2451) within hydrogen bonding distance ofa non-bridging oxygen of the phosphoramide (see FIG. 10(B)). Theinhibitor's two C's, which correspond to C74 and C75 of peptidyl-tRNA,are Watson-Crick base-paired with G2285 (2252) and G2284 (2251)in theP-loop, respectively (see FIG. 11(A)). The C74-G2285 (2252) interactionwas predicted by the results of Noller and coworkers (Noller et al.(1992) Science 256:1416-1419). The dA, which corresponds to A76 of atRNA in the P-site, is not base-paired, but rather stacks on the riboseof A2486 and hydrogen bonds to the 2′OH of nucleotide A2485 (see FIG.12(B)).

[0204] Only the CC-puromycin moiety of the mini-helix acceptor stemanalogue showed ordered electron density in its difference electrondensity map (see FIG. 10(C)). The C75 of the acceptor stem CCA isWatson-Crick base-paired with G2588 (2553) of the A-loop, whereas theC74 is more disordered and not base-paired but appears to stack on aribosome base. The dimethyl A of the A-site inhibitor puromycin ispositioned identically to the dimethyl A of the Yarus inhibitor.Further, the dimethyl A of puromycin, which is the A76 equivalent of anA-site tRNA, interacts with the A-loop in much the same way that the A76from the P-site CCA interacts with the P-loop (see FIG. 11(B)).

[0205] The most notable of the several conformational changes in theribosome induced by the binding of the transition state analogue is theordering of base A2637 (2602), which is disordered in the unligandedenzyme (see FIG. 11(B)). It becomes positioned between the CCA bound atthe A-site and the CCA bound at the P-site. The base of U2620 (2585)also moves so that it can make a hydrogen bond with the 2′ hydroxyl ofthe ribose of A76 in the A-site, and U2619 and G2618 shift to allow theA76 to be positioned. Smaller shifts in positions are observed in thepositions of A2486, whose N3 is near to the non-bridging oxygen of thephosphate, and one of the G residues with which it interacts, G2102(2482).

[0206] 2. Location and Chemical Composition of the Peptidyl TransferaseSite.

[0207] The inhibitors are bound to a site made entirely of 23S rRNA withno proteins nearby, proving that the ribosome is a ribozyme. Both theYarus inhibitor and the A-site analogue of aa-tRNA bind to the largesubunit at the bottom of a large and deep cleft at the entrance to the100 Å long polypeptide exit tunnel that runs through to the back of thesubunit (see FIG. 12). This site is surrounded by nucleotides belongingto the central loop of 23S RNA domain V, the “peptidyl transferaseloop.” Nucleotides from the single stranded portions of this loop makethe closest approach to the phosphate that mimics the tetrahedral carbonintermediate. In general, the helices that extend from the peptidyltransferase loop in 2 secondary structure diagrams of 23S rRNA alsoextend away from the active site in the tertiary structure (see FIG.13). Although there are 13 proteins that interact with domain V (seeFIG. 14(A)), there are no globular proteins in the vicinity of theinhibitor. The closest polypeptides are the non-globular extensions ofseveral proteins (L2, L3, L4, L10e) that penetrate deeply into domain Vand approach the active site (see FIG. 14(B)). These extensions fillmany of the voids between the RNA helices of domain V, neutralizephosphate backbone charge, and presumably stabilize the structure of thedomain and its association with other RNA regions. However, none oftheir side chain atoms is closer than about 18 Å to the phosphorus ofthe inhibitor's phosphate group, which marks the site where peptidebonds form. Furthermore, the substrate analogue is completely enclosedin an rRNA cavity that is so tightly packed that there is no possibilitythat an unidentified peptide could be lurking nearby (see FIG. 15).Thus, the catalytic entity in the ribosome must be RNA.

[0208] Two of the proteins with long termini or loops penetrating therRNA scaffold of domain V are proteins that could not previously beexcluded from involvement in the peptidyl transferase reaction L2 and L3(Noller (1991) Ann. Rev. Biochem. 60: 191-227). Noller and colleagues(Noller et al. (1992) supra) found that under conditions which preventRNA denaturation, extensive digestion of Thermus thermophilus 50Ssubunits with proteases followed by extraction with phenol and otheragents that disrupt protein-RNA interactions did not remove severalpeptides from the subunit that were less than 10,000 in molecularweight. The structure makes it clear why these protein fragments wereparticularly resistant to protease treatments. While protease treatmentcould digest the globular protein domains on the surface of the largesubunit, it could not remove the long termini or loops that penetratedeeply in the 23S rRNA because they are sequestered within the rRNA andthus protected from cleavage, independently of the globular domains.

[0209] 3. Peptidyl Transferase Active Site.

[0210] The RNA that surrounds the substrate analogues is closely packed,much like the active site region of a protein enzyme and the nucleotidesin contact with the inhibitor are greater than 95% conserved in allthree kingdoms of life (see FIG. 15). Thus, it is clear that theribosome is a ribozyme, but what gives the RNA its catalytic power?

[0211] Without wishing to be bound by theory, the residue most likely tobe involved in catalysis, presumably as a general base, is A2486, whoseN3 is about 3 Å from the phosphoramide oxygen of the Yarus inhibitorthat is the analogue of the carbonyl oxygen of a nascent peptide bondand about 4 Å from the amide that corresponds to the amide nitrogen ofthe peptide bond being synthesized. Ordinarily, the pKa of the N1 ofadenosine monophosphate is about 3.5 and that of its N3 is perhaps 2 pHunits lower (Saenger (1984) Principles of Nucleic Acid Structure, (C. R.Cantor, eds.), Springer Advanced Texts in Chemistry, Springer-Verlage,New York, N.Y.), and in order for A2486 to function as a general base,its pKa would have to be raised to 7 or higher. The crystal structureitself suggests that its pKa is, in fact, quite unusual. The N3 of A2486can only hydrogen bond to the phosphate oxygen, as observed, if it (orthe phosphate oxygen) is protonated. The distance between these twoatoms is about 3 Å indicating that a hydrogen bond does, indeed, existbetween them. Since the crystal is at pH 5.8, this implies that the pKaof the N3 is greater than 6. Muth and Strobel have measured the pKa ofthe corresponding A in E. coli 23S RNA by examining its dimethyl sulfatereactivity as a function of pH and have concluded that it is 7.6, thoughthey cannot be sure from their experiments whether it is the N3 or N1whose pKa they have measured (Muth et al. (2000) Science 289: 947-950).Because there is no other available, titratable RNA functional groupcloser than about 7 Å to the nascent peptide bond, there is not othergroup available to function as a general base.

[0212] There are several features of environment of A2486 (2451) thatmight affect its pKa. The pKa of the N3 of A2486 (2451) may be increasedsignificantly in part by a charge relay mechanism, analogous to thatwhich occurs in the active site of the serine proteases, with the buriedphosphate of A2485 (2450) performing a similar function as the buriedcarboxylate of Asp102 of chymotrypsin. The experimental 2.4 Å electrondensity map unambiguously establishes the hydrogen bonding interactionsin this most critical region of the active site (see FIG. 16(A)). The N6of A2486 interacts with the O6 atoms of G2482 (2447) and G2102 (2061)(see FIG. 16(B)). The N2 of G2482 is also interacting with anon-bridging oxygen of the phosphate group of A2485 (2450) that is amongthe total of 3 most solvent inaccessible phosphate groups (826, 1497 and2485) in the large ribosomal subunit for which we do not see anyneutralizing counterion in the 2.4 Å resolution map. Weak density thatmay correspond to a water molecule is hydrogen bonded to the othernon-bridging oxygen. A neutralizing counterion is not apparent in thisstructure. The buried phosphate of A2485 could abstract the proton fromthe exocyclic N2 of G2482 in order to neutralize its energeticallyunfavorable buried negative charge. This, in turn, would stabilize theotherwise rare imino tautomer of that base. The interaction of an iminoof G2482 with A2486 likewise can stabilize the imino tautomer of A2486that would result in a negative charge on its N3 were it unprotonated(see FIG. 16(C)). In this way, some of the negative electrostatic chargeoriginating on the buried phosphate of A2485 could be relayed to the N3of A2486, thereby increasing its pKa.

[0213] A second feature of the environment of the catalytic site thatmay affect its stability, tautomeric state and electrostatic chargedistribution is a bound monovalent cation. A potassium or a sodium ioninteracts with the O6 of G2482 and G2102 as well as with three otherbases. Its identity as a potassium ion is established by its observedcontinuation and by an independent experiment showing that a rubidiumion can bind to this site. The monovalent ion might also stabilizenon-standard tautomers, but its expected influence on the pKa of A2486is less obvious. Early biochemical experiments have shown the importanceof potassium for peptidyl transferase activity (Monro (1967) supra;Maden et al. (1968) supra) and this binding site could be responsiblefor that affect.

[0214] It may also be the case that stabilization of an imino tautomerby a buried phosphate explains the expected higher pKa of a catalyticcytosine in the active site of the hepatitis delta virus ribozyme(Ferre-D'Amare et al. (1998) Nature 395: 567-574; Naharo et al. (2000)Science 287: 1493-1497). In this case, a backbone phosphate, whosesolvent accessibility is similar to that of A2485 in the ribosome, isobserved to hydrogen bond to the N4 of C, and the protonated form of theimino tautomer of that C would neutralize the phosphate, promoting thefunction of its N3 as a general acid (Naharo et al. (2000) supra).

[0215] 4. Catalytic Mechanism of Peptide Bond Formation.

[0216] The proximity of the N3 of A2486 (2451) to the peptide bond beingsynthesized and the nature of the reaction catalyzed suggest a chemicalmechanism of peptide synthesis that is analogous to the reverse of thedeacylation step seen in serine proteases during peptide hydrolysis(Blow et al. (1969) Nature 221: 337-340; Steitz et al. (1982) Ann. Rev.Biophys. Bioeng. 11: 419-444). In that mechanism, the basic form ofHis57 abstracts a proton from the α-amino group of the peptide productas it attacks the acyl-Ser195. Formation of the tetrahedral carbonylcarbon intermediate is stabilized by interaction of the oxyanion formedwith backbone amides (the “oxyanion hole”); His57 shuttles the protonacquired from the α-NH₂ to Ser195 as the tetrahedral intermediate breaksdown.

[0217] The residue A2486 (2451) appears to be the analogous to His57 inchymotrypsin and that the peptidyl-tRNA is analogous to acyl-Ser195.Thus, the N3 of A2486, with its greatly elevated pKa, abstracts a protonfrom the α-amino group of the A-site bound aminoacyl-tRNA facilitatingthe nucleophilic attack of that amino group on the carbonyl carbon thatacylates the 3′ OH of the tRNA in the P-site (see FIG. 17(A)). Incontrast to the serine proteases, however, the oxyanion of thetetrahedral intermediate is near to the protonated N3 of A2486 (A245 1)rather than being proximal to a separate oxyanion binding site. Thus, itcould be that the protonated N3 of A2486 stabilizes the formation of theoxyanion by hydrogen bonding to it, as we observe in the Yarus inhibitorcomplex (see FIG. 17(B)). The N3 of A2486 could then subsequentlytransfer its proton to the 3′ hydroxyl of the P-site bound tRNA, whichis liberated as the peptide shifts to the A-site bound tRNA (see FIG.17(C)).

[0218] An additional question is how is the catalyzed hydrolysis of thepeptidyl tRNA in the P-site prevented prior to the delivery of the nextappropriate aa-tRNA to the A-site? It appears from this complex thatwater would not be excluded from access to the peptidyl link to theP-site tRNA if the A-site were vacant. An analogous problem wasdiscussed by Koshland in the 1960s (Koshland, Jr. (1963) Cold SpringHarbor Symp. Quant. Biol. 28: 473-489), who theorized why hexokinasedoes not hydrolyze ATP in the absence of glucose, since water shouldbind perfectly well to the binding site used by the 6-hydroxyl ofglucose. The answer proposed was induced fit, i.e., hexokinase is notcatalytically competent until the glucose binds and produces aconformational change that orients substrates and catalytic groupsoptimally. This indeed appears to be the case (Bennett, Jr. et al.(1978) Proc. Natl. Acad. Sci. USA 75: 4848-4852). Similarly, it could bethat the catalytic A2486 and/or the peptidyl substrate are not properlyoriented or that the binding site for the α-NH₂ group is blocked by areoriented ribosome base in the absence of aa-tRNA in the A-site. We doobserve that the base of U2620 is close to A2486 in the ligand freestructure, and it may serve as the appropriate plug that preventsspontaneous hydrolysis of peptidyl-tRNA.

[0219] Thus, it appears that this RNA enzyme uses the same principles ofcatalysis as a protein enzyme. First, a large catalytic enhancement isachieved by precisely orienting the two reactants, the αNH₂ from theA-site aminoacyl-tRNA and the carbonyl carbon from the P-sitepeptidyl-tRNA. This may be accomplished, in part, by the interactions ofthe CCA ends of the A-site and P-site tRNAs with the A-loop and P-loop,respectively. Secondly, acid-base catalysis and transition statestabilization are achieved by an enzyme functional group (A2486 (2451)in this case) whose chemical properties are altered appropriately by theactive site environment. Third, similar chemical principles may be usedby RNA and protein enzymes to alter the pKa's of functional groups. Aburied carboxylate of Asp102 acting through His57 alters thenucleophilicity of Ser195 in chymotrypsin (Blow et al. (1969) supra). Inthe ribosome a solvent inaccessible phosphate may act through G2482(2447) alters the nucleophilicity of the N3 of A2486 (2451). It could bethat RNA molecules “learned” how to use the chemical principles ofcatalysis significantly before protein molecules did.

[0220] 5. tRNA Binding.

[0221] While it is not possible to bind tRNA molecules to either theA-or P-sites in these crystals for steric reasons, it is possible toplace the A-, P- and E-site tRNA molecules on the large ribosomalsubunit in the same relative orientation that Cate et al. observed intheir crystallographic study of the Thermus aquaticus 70S ribosome. Theco-ordinates of the three tRNA molecules in the relative positions seenin the 70S ribosome can be docked on the Haloarcula marismortui largeribosomal subunit in a way that avoids steric clashes and places theacceptor stems of the A-site and P-site tRNAs near to the positions ofthe CCAs we have observed bound to the A-loop and P-loop (see FIG. 18).Although nucleotides C74 and C75 were modeled in a differentconformation in the 7.8 Å ribosome map, the C74 residues from the CCAsin both the A- and P-sites can be connected to residue 72 of the dockedA-site and P-site tRNAs through a modeled residue 73, and it appearsthat the tRNA molecules fit well onto the surface of the subunit.Unexpectedly, this modeling places the E-site, P-site and A-site boundtRNA molecules in close proximity to three ribosomal proteins. ProteinsL5 and L10e are near tRNAs in the P-site and A-site. Since both of theseproteins also interact with 5S rRNA, this observation raises thepossibility that 5S rRNA and some of its associated proteins might helpstabilize the positioning of ribosome bound tRNAs and is consistent withthe fact that 5S rRNA enhances ribosomal activity, but is not absolutelyessential for it (Moore, Ribosomal RNA & Structure, Evolution,Processing and Function in Protein Biosynthesis (1996), supra, pp.199-236). Protein L44e appears to interact with the E-site tRNA and maycontribute to E-site activity. According to this docking experiment theA-site tRNA interacts with the highly conserved stem-loop 2502-2518(2467-2483) which together with L10e forms a large concave surface thatcontacts the tRNA on the T-stem, utilizing the exact same binding siteexploited by EF-Tu (Gutell et al. (2000) supra).

[0222] Examination of the relationships between the CCAs bound in the A-and P-sites and the tRNAs to which they are connected as well as theirinteractions with the ribosome also leads to some insights intotranslocation. Immediately after formation of the new peptide bond anddeacylation of the P-site tRNA, the acceptor end of the P-site tRNA isknown to move to the E-side and that of the A-site tRNA moves to theP-site (Blobel et al. (1970) J. Cell. Biol. 45: 130-145). Theapproximate modeling of the 3 tRNA molecules on the large subunitsuggests some possible contributions to this process. First, there aretwo base-pairs between the P-site tRNA and the P-loop and only onebetween the A-site and the A-loop. Moving from the A- to the P-siteincreases base-pairing, though there must be a concomitant attraction ofthe deacylated P-site tRNA to an E-site. Further, the CCAs bound to theA and P loops are related by 180° rotation, whereas the tRNAs to whichthey are attached are not. Thus, the relationships of the CCAs to theacceptor stems cannot be the same in both sites and may not be equallystable. If the conformation of the A-site tRNA is less stable, thenmoving a tRNA from the A- to the P-site might be energetically favored.

[0223] 6. Polypeptide Exit Tunnel.

[0224] It appears very likely from the structure that all nascentpolypeptides pass through the polypeptide exit tunnel before emergingfrom the ribosome, because there appears to be no other way out. We arenow able to address two important questions about the functioning of thepolypeptide exit tunnel: (1) Why do nascent proteins not stick to itswalls? Teflon has the marvelous property of not sticking to denaturedegg proteins, so how has the ribosome achieved a similar non-sticksurface for the denatured proteins that must pass through the tunnel?(2) Do proteins fold to any degree in the tunnel giving the ribosome achaperon-like function?

[0225] The length of the tunnel from the site of peptide synthesis toits exit is about 100 Å, broadly consistent with the length of nascentpolypeptide that is protected from proteolytic cleavage by the ribosome(Moazed et al. (1989) Nature 342:142) and the minimum length requiredfor antibody recognition at the exit (Picking et al. (1992) Biochemistry31: 2368-2375). The tunnel is largely straight, except for a bend 20 to35 Å from the peptidyl transferase center (see FIG. 19). Its diametervaries from about 20 Å at its widest to a narrow point of about 10 Å atthe very beginning and at a position 28 Å from the tunnel exit with anaverage diameter of about 15 Å. Since the smallest orifice through whichthe polypeptide product must pass only barely accommodates the diameterof an α-helix diameter, it seems unlikely that significant proteinfolding beyond the formation of α-helix could occur within the ribosome.

[0226] The majority of the tunnel surface is formed by domains I-V of23S rRNA, but significant contributions are also made by thenon-globular regions of proteins L22, L4 and L39 which not only fillsome of the voids in the RNA scaffold, but also form significantportions of the tunnel wall (see FIG. 19). The largest proteincontributor to the surface of the tunnel is L22 whose long α-hairpinloop lies between RNA segments of domains I through IV and isapproximately parallel with the axis of the tunnel. Unlike the othertunnel proteins, protein L39 does not have a globular domain at thesurface of the particle and is almost entirely buried in domains I andIII underneath protein L23. Interestingly, the nucleotides of 23S rRNAthat form the tunnel wall are predominantly from loops in the 23S rRNAsecondary structure (see FIG. 19). As it progresses through the tunnelfrom the active site, a nascent polypeptide first encounters domain Vfollowed 20 Å further along by domains II and IV and proteins L4 andL22. The last half of the tunnel is formed by domains I and III and theprotein L39e.

[0227] The narrowest part of the tunnel is formed by proteins L22 and L4which approach the tunnel from opposite sides forming what appears to bea gated opening (see FIG. 19C). The function of this constriction, ifany, is not obvious. It might be the place where the nature of thenascent chain is sensed and the information transmitted to the surfaceof the particle, perhaps through L22 or L4. The α-hairpin of L22 at thesite of this orifice and the 23S rRNA interacting with it are highlyconserved; its globular portion is located adjacent to the tunnel exiton the surface that must face the translocon during protein secretion(see FIG. 19).

[0228] The “non-stick” character of the tunnel wall must reflect a lackof structural and polarity complementarity to any protein sequence orconformation that it encounters. The tunnel surface is largelyhydrophilic and includes exposed hydrogen bonding groups from bases,backbone phosphates and polar protein side-chains (see FIG. 19). Whilethere are many hydrophobic groups (sugars, bases, protein side-chains)facing the tunnel as well, there are no patches of hydrophobic surfacelarge enough to form a significant binding site for hydrophobicsequences in the nascent polypeptide. As the tunnel is some 20 Å indiameter and filled with water and the newly synthesized polypeptide ispresumably freely mobile, the binding of a peptide to the tunnel wallwould result in a large loss of entropy that would have to becompensated for by a large complementary interaction surface that islarger than 700 Å (Chothia et al. (1975) Nature 256: 705-708).Similarly, while Arg and Lys side-chains from a nascent peptide mayindeed interact with the phosphates exposed in the tunnel, the degree ofstructural complementarity and the net binding energy obtained afterdisplacing bound counterions must be too small to overcome the largeunfavorable entropy of immobilization that would result from peptidebinding. Thus, although the ribosome tunnel is made primarily of RNA,the nature of its surface is reminiscent of the interior surface of thechaperonin, GroEL (Xu et al. (1998) J. Struct. Biol. 124:129-141) in itsnon-binding conformation. Only in the conformation that exposes a largehydrophobic surface does GroEL bind a denatured protein.

[0229] There are six proteins (L19, L22, L23, L24, L29 and L31e) locatedat the exit from the tunnel, facing the translocon onto which theribosome docks during protein secretion. There is evidence that theribosome binds the translocon even after extensive digestion of itsprotein by protease implying that interaction between the translocon andthe ribosome is mediated by RNA. The proximity of these proteins to thetranslocon, however, leads us to wonder what role, if any, they mightplay in the protein secretion process. Recent data from the Dobbersteinlaboratory shows that the N-terminal domain of SRP54, the G-protein fromthe signal recognition particle involved in signal peptide binding, canbe crosslinked to ribosomal proteins L23 and L29. These two proteins areadjacent to each other and at the tunnel exit (see FIG. 19).

[0230] 7. Evolution.

[0231] In vitro evolution of RNA oligonucleotides has produced small RNAmolecules that can bind molecules like the Yarus inhibitor effectivelyor catalyze the peptidyl transfer reaction (Zhaug et al. (1998) Chem.Biol. 5: 539-553; Welch et al (1997) supra). The sequence and secondarystructure of one of these selected RNAs is reminiscent of the peptidyltransferase loop in domain V of 23S rRNA (Zhaug et al. (1998) supra).The most striking similarity is a five nucleotide sequence that isidentical to a sequence in domain V that includes the catalytic A2486,G2482 and the buried phosphate of A2485. Remarkably, all of the groupsinvolved in the proposed charge relay system for activating A2486 in theribosome, are present in the in vitro selected ribozyme. Thus, thoughthe surrounding structural context is likely to be different, it seemsplausible that this artificially evolved ribozyme uses the samemechanisms as the ribosome for shifting the pKa of an adenine andlikewise uses it as a base for peptide synthesis. A second RNA (Welch etal. (1997) supra) contains a 12 nucleotide loop that includes a 9-basesequence identical to that found in the same region of the peptidyltransferase loop.

[0232] The striking similarities between the sequences containing thekey catalytic elements found in the peptidyl-transferase active site ofthe ribosome and sequences of in vitro selected RNAs having relatedactivities make it clear that the appearance of a small RNA domaincapable of catalyzing peptidyl transferase was a plausible first step inthe evolution of protein synthesis on the ribosome. The first peptidessynthesized by this primordial peptide synthesizing enzyme might havebeen random polymers or copolymers, and it may have functioned withsubstrates as simple as an aminoacylated CCA. Basic peptides of thetypes observed to form the non-globular extensions that co-fold with the23S rRNA might have been among the first peptides synthesized that werefunctionally useful. Such peptides may have enhanced the stability ofthe protoribosome and other early ribozymes as the more sophisticatedpeptides of the present day ribosome appear to do.

[0233] C. Atomic Structure of the Large Ribosomal Subunit at 2.4 ÅResolution, Complete Refinement

[0234] The three-dimensional structure of the large ribosomal subunitfrom Haloarcula marismortui has now been completely refined at 2.4 Åresolution. The model includes 2876 RNA nucleotides, 3701 amino acidsfrom 28 ribosomal proteins, 117 magnesium ions, 88 monovalent cations,and 7898 water molecules. Many of its proteins consist of asurface-exposed globular domain and one or more basic, non-globularextensions that are buried in rRNA. Half of them include motifs commonin non-ribosomal proteins including, for example: RRM domains, SH3-likebarrels and zinc fingers. Proteins that have significant sequence andstructural similarity, such as L15 and L18e, make essentially identicalinteractions with rRNA.

[0235] More particularly, the H. marismortui 50S subunit has beencompletely rebuilt and refined by successive rounds of gradient energyminimization and B-factor refinement using CNS (Brünger et al. (1998)supra). Ribosomal proteins and rRNA were completely rebuilt using thesoftware program “O” (Jones, T. A. et al. (1991) Acta Crystallogr. A46:110-119) with 2F_(o)-Fc electron density maps prior to the modeling ofsolvent and metal ions. Modeling errors in the proteins were identifiedusing PROCHECK (Laskowski et al.(1993) J. Appl. Cryst. 26: 283-291) andby inspection of F_(o)-Fc maps. Difference maps also aided in theidentification of errors in the rRNA, most often associated with sugarpuckers. In the process, some adjustments were made in amino acidconformations, sequence register and in sequences themselves. Sequencechanges made were largely limited to L10e, L15e, and L37Ae, the onlythree proteins from the H. marismortui 50S that have not been sequenceddirectly. In addition, fifty-one amino acids were added to the modeldescribed in section IIA with forty-four of these coming from L10 at thebase of the L7/L12 stalk and L39e which lines a portion of the wall ofthe polypeptide exit tunnel. Fewer adjustments were made to the rRNAstructure. Forty-nine new nucleotides were modeled and refined, mainlyin helices 43 and 44 in domain II of 23S rRNA. In addition, the sugarpucker or conformation about the glycosidic bond was adjusted for somenucleotides. The refinement process was monitored by the quality ofelectron density maps calculated using phases derived from the model aswell as R/R_(free) values. The completely refined model now includes2876 RNA nucleotides, 3701 amino acids, 210 metal ions, and 7898 watermolecules. The model refines to an R/R_(free) of 18.9% /22.3% and hasexcellent geometry (Table 4).

[0236] Solvent modeling began with the generation of a list of possiblemagnesium ions obtained by an automatic peak selection using CNS. Peaksgreater than 3.5σ in F_(o)-Fc maps positioned within the magnesiuminner-sphere coordination distance of 1.9-2.1 Å from N or O atoms wereselected. The resulting list was manually inspected and only peaks thatdisplayed clear octahedral coordination geometry were selected asmagnesium.

[0237] Monovalent cations were identified on the basis of isomorphousdifferences between rubidium-soaked and native crystals of the H.marismortui 50S subunit. Since native crystals were stabilized in thepresence of 1.6M NaCl, these sites were initially modeled and refined asNa⁺¹. Refinement of these sites as K⁺¹ almost always resulted inunusually high temperature factors, with two exceptions where we havemodeled K⁺¹. Most of the monovalent sites in the 50S subunit appear tooccupied by Na⁺¹ in our crystals, however, these sites are likely to beoccupied by K⁺¹ in vivo.

[0238] Waters were selected as peaks greater than 3.5σ in F_(o)-Fcelectron density maps and between 2.5 and 3.3 Å of O or N atoms.Individual B-factor values were used to assess the assignment of watermolecules. A number of waters refined to B-factors significantly lowerthan surrounding RNA and protein atoms. In many cases these peaks werefound to be metal or chloride ions. A small number of low B-factor watermolecules were retained in the final model because they could not beunambiguously assigned as other species. As a result of adding metalions and water molecules the final model now contains 98, 547non-hydrogen atoms. The refinement and model statistics for the largeribosomal subunit are summarized in Table 4. TABLE 4 Refinement andModel Statistics for the H marismortul 50S Subunit Space Group C222₁ a =211.66Å, b = 299.67Å, c = 573.77Å Total non-hydrogen atoms 98,542 RNAatoms 61,617 Protein atoms 28,800 Water molecules 7,893 Magnesium ions117 Potassium ions 2 Sodium ions 86 Chloride ions 22 Cadmium ions 5Refinement Statistics: Resolution Range 15.0 − 2.4 Å Number ofreflections used in refinement 623,525 Number of reflections forcross-validation 6,187 R_(working) 18.9% R_(free) 22.3% σ_(a) coordinateerror (cross-validated) 0.35Å(0.43Å) luzzati coordinate error(cross-validated) 0.29Å(0.35Å) Deviations from ideality: r.m.s.d. bondlengths 0.0052Å r.m.s.d. bond angles 1.13° r.m.s.d. dihedrals 15.7°r.m.s.d. impropers 2.12° Protein Statistics from Ramachandran Plot:Residues in most favored regions 2704 (86.6%) Residues in additionalallowed regions 379 (12.1%) Residues in generously allowed regions 27(0.9%) Residues in disallowed regions 13 (0.4%) Average B-factorStatistics (Å²): All atoms (high/low) 44.3 (10.1/133.7) rRNA 41.2(11.78/125.0) proteins 49.7 (13.9/92.5) waters 41.89 (9.58/115.4)

[0239] Refinement has also permitted additional modeling of L10, L39e,and the L11 binding site in 23S rRNA. Furthermore, it has beendiscovered that certain motifs, for example, RRM topologies, SH3-likebarrels and zinc fingers are common in the 50S proteins and eachrecognizes rRNA in many different ways. Proteins that have significantthree-dimensional homology, however, such as L15 and L18e as well as L18and S11, make essentially identical interactions with rRNA. Additionalstructural homologies between 50S proteins and non-ribosomal proteinsalso are apparent. The solvent exposed surfaces of these globularprotein domains are rich in aspartate and glutamate residues, whileirregular protein extensions penetrate the RNA core of the ribosome.These extensions are often highly conserved, and their abundance ofarginine, lysine, and glycine residues is important for their function.Collectively, the results show evolutionary connections between manyribosomal proteins and illustrate that protein-RNA interactions in theribosome, although largely idiosyncratic, share some common principles.

[0240] D. Antibiotic Binding Sites

[0241] In addition to the foregoing structural studies, the structure ofthe large ribosomal subunit of H. marismortui has been determinedcomplexed with each of seven different antibiotics. More specifically,crystals of the H. marismortui large ribosomal subunit have been soakedwith one of the following antibiotics: anisomycin, blasticidin,carbomycin, tylosin, sparsomycin, virginiamycin or spiramycin. Thestructure of the large ribosome subunit complexed with each antibioticwas then resolved based on X-ray diffraction data generated for eachcrystal.

[0242] Briefly, a small amount of a concentrated antibiotic solution wasadded to a large subunit crystal suspended in stabilization solution andincubated for several hours. Following freezing and the other proceduresnormally used to prepare such crystals for experimental use, X-raydiffraction data were collected from the antibiotic containing crystals.Because the crystals were isomorphous to those from which the structuredescribed above was derived, the phases obtained for native crystal werecombined with the diffraction intensities obtained from theantibiotic-soaked crystal to obtain a structure for the latter. Theposition of the antibiotic in the crystal to which was bound is revealedmost clearly in difference electron density maps, which are electrondensity maps computed using the phases just referred to and amplitudesobtained by subtracting the amplitudes of crystals that contain noantibiotic from the (suitably scaled) amplitudes of those that containantibiotic. By using the foregoing methods, it was possible to determinethe atomic co-ordinates that show the spatial relationship betweenparticular antibiotics and their binding sites within the largeribosomal subunit. It is contemplated that similar methods can be usedto resolve the structure of other antibiotics complexed to the largeribosomal subunit.

[0243] The atomic co-ordinates of the large ribosomal subunit complexedwith anisomycin are listed in a table on compact disk Disk No. 3 of 3under the file name anisomycin.pdb. In addition, FIG. 20 shows thespatial relationship between the antibiotic anisomycin and the largeribosomal subunit.

[0244] The atomic co-ordinates of the large ribosomal subunit complexedwith blasticidin are listed in a table on compact Disk No. 3 of 3 underthe file name blasticidin.pdb. FIG. 21 shows the spatial relationshipbetween the antibiotic blasticidin and the large ribosomal subunit. Fororientation, FIG. 21 also includes a substrate for the P-site.

[0245] The atomic co-ordinates of the large ribosomal subunit complexedwith carbomycin are listed in a table on compact disk Disk No. 3 of 3under the file name carbomycin.pdb. FIG. 22 shows the spatialrelationship between the antibiotic carbomycin and the large ribosomalsubunit. FIG. 22 also shows a portion of the polypeptide exit tunnel.

[0246] The atomic co-ordinates of the large ribosomal subunit complexedwith tylosin are listed in a table on compact disk Disk No. 3 of 3 underthe file name tylosin.pdb. FIG. 22 shows the spatial relationshipbetween the antibiotic tylosin and the large ribosomal subunit. FIG. 22also shows a portion of the polypeptide exit tunnel.

[0247] The atomic co-ordinates of the large ribosomal subunit complexedwith sparsomycin are listed in a table on compact disk Disk No. 3 of 3under the file name sparsomycin.pdb. FIG. 23 shows the spatialrelationship between the antibiotic sparsomycin and the large ribosomalsubunit. For orientation, FIG. 23 also shows a substrate for the P-site.

[0248] The atomic co-ordinates of the large ribosomal subunit complexedwith virginiamycin are listed in a table on compact disk Disk No. 3 of 3under the file name virginiamycin.pdb. FIG. 24 shows the spatialrelationship between the antibiotics virginiamycin as well carbomycin,and the large ribosomal subunit.

[0249] The atomic co-ordinates of the large ribosomal subunit complexedwith spiramycin are listed in a table on compact disk Disk No. 3 of 3under the file name spiramycin.pdb.

[0250]FIG. 25 shows the spatial orientations of several antibiotics,namely, blasticidin, anisomycin, virginiamycin and carbomycin, as theybind to their respective antibiotic binding sites within the largeribosomal subunit. For purposes of orienting the reader, the positionsof the P-site, A-site and the polypeptide exit tunnel are shown in FIG.25. As is apparent, these antibiotics bind to or contact specificlocations within the large ribosomal subunit to disrupt proteinbiosynthesis. For example, it appears that blasticidin binds the largeribosomal subunit in the vicinity of the P-site; anisomycin andvirginiamycin bind the large ribosomal subunit in the vicinity of theA-site; and carbomycin (a macrolide) binds the large ribosomal subunitin the vicinity of the polypeptide exit tunnel adjacent the peptidyltransferase site.

[0251] From FIG. 25, it is apparent that the skilled artisan mayidentify certain portions of each antibiotic that contact regions in thelarge ribosomal subunit. By knowing their spatial relationship withrespect one another, the skilled artisan may generate a hybridantibiotic molecule comprising a portion of a first template antibioticand a portion of a second, different template antibiotic. The twoportions may be linked by a chemical linker so as to maintain thespatial orientation of one portion with respect to the other portion. Asa result, the hybrid antibiotic may simultaneously bind each of theregions of the ribosomal subunit typically bound by each templateantibiotic. The design and testing of such molecules is discussed inmore detail below.

[0252] E. Experimental Techniques Which Exploit X-Ray Diffraction Data

[0253] Based on the X-ray diffraction pattern obtained from theassemblage of the molecules or atoms in a crystalline solid, theelectron density of that solid may be reconstructed using tools wellknown to those skilled in the art of crystallography and X-raydiffraction techniques. Additional phase information extracted eitherfrom the diffraction data and available in the published literatureand/or from supplementing experiments may then used to complete thereconstruction.

[0254] For basic concepts and procedures of collecting, analyzing, andutilizing X-ray diffraction data for the construction of electrondensities see, for example, Campbell et al. (1984) BiologicalSpectroscopy, The Benjamin/Cummings Publishing Co., Inc., (Menlo Park,Calif.); Cantor et al. (1980) Biophysical Chemistry, Part II: Techniquesfor the study of biological structure and function, W. H. Freeman andCo., San Francisco, Calif.; A. T. Brünger (1993) X-PLOR Version 3.1: Asystem for X-ray crystallography and NMR, Yale Univ. Pr., (New Haven,Conn.); M. M. Woolfson (1997) An Introduction to X-ray Crystallography,Cambridge Univ. Pr., (Cambridge, UK); J. Drenth (1999) Principles ofProtein X-ray Crystallography (Springer Advanced Texts in Chemistry),Springer Verlag; Berlin; Tsirelson et al. (1996) Electron Density andBonding in Crystals: Principles, Theory and X-ray DiffractionExperiments in Solid State Physics and Chemistry, Inst. of Physics Pub.;U.S. Pat. No. 5,942,428; U.S. Pat. No. 6,037,117; U.S. Pat. No.5,200,910and U.S. Pat. No. 5,365,456 (“Method for Modeling the Electron Densityof a Crystal”).

[0255] A molecular model may then be progressively built using theexperimental electron density information and further refined againstthe X-ray diffraction data resulting in an accurate molecular structureof the solid.

[0256] F. Structural Determination Of Other Large Ribosomal Subunits

[0257] It is understood that the skilled artisan, when provided with theatomic co-ordinates of a first macromolecule may use this information toquickly and easily determine the three-dimensional structure of adifferent but structurally related macromolecule. For example, theatomic co-ordinates defining the large ribosomal subunit from H.marismortui can be used to determine the structure of the largeribosomal subunit from other species either as an isolated subunit, incomplex with the small subunit, or either of these complexed withfunctionally important ligands, for example: aminoacyl tRNA; variousprotein synthesis factors, such as elongation factor G, elongationfactor Tu, termination factor or recycling factor, in both their GTP andGDP conformational states; and protein synthesis inhibitors, forexample, antibiotics. In addition, the H. marismortui subunitco-ordinates can also be used to solve the structures of ribosomalcomplexes with components of the protein secretion machinery, forexample, the signal recognition particle, and the translocon.

[0258] If the crystal being examined contains a macromolecule of unknownstructure and no additional information is available, additionalexperiments sometimes may be required to determine the relevant phasesof the macromolecule. These studies can often be time consuming anduncertain of success (Blundell et al. (1976) supra). However, whenadditional information, for example, structural and/or crystallographicinformation, is available for molecules related in some way to themacromolecule of interest then the process of resolving the structure ofthe molecule of interest is a much less challenging and time-consumingtask.

[0259] Accordingly, the skilled artisan may use information gleaned fromthe prior resolved structure to develop a three-dimensional model of anew molecule of interest. Furthermore, the skilled artisan may use avariety of approaches to elucidate the three-dimensional structure ofthe new molecule. The approaches may depend on whether crystals of themolecule of interest are available and/or whether the molecule ofinterest has a homologue whose structure has already been determined.

[0260] In one approach, if the molecule of interest forms crystals thatare isomorphous, i.e., that have the same unit cell dimensions and spacegroup as a related molecule whose structure has been determined, thenthe phases and/or co-ordinates for the related molecule can be combineddirectly with newly observed amplitudes to obtain electron density mapsand, consequently, atomic co-ordinates of the molecule of interest. Theresulting maps and/or atomic co-ordinates may then be refined usingstandard refinement techniques known in the art. In another approach, ifthe molecule of interest is related to another molecule of knownthree-dimensional structure, but crystallizes in a different unit cellwith different symmetry, the skilled artisan may use a technique knownas molecular replacement to obtain useful phases from the co-ordinatesof the molecule whose structure is known (Blundell et al. (1976) supra).These phases can then be used to generate an electron density map and/oratomic co-ordinates for the molecule of interest. In another approach,if no crystals are available for the molecule of interest but it ishomologous to another molecule whose three-dimensional structure isknown, the skilled artisan may use a process known as homology modelingto produce a three-dimensional model of the molecule of interest. It iscontemplated that other approaches may be useful in deriving athree-dimensional model of a molecule of interest. Accordingly,information concerning the crystals and/or atomic co-ordinates of onemolecule can greatly facilitate the determination of the structures ofrelated molecules.

[0261] The method of molecular replacement, developed initially byRossmann and Blow in the 1960s, is now used routinely to establish thecrystal structures of macromolecules of unknown structures using thestructure of a homologous molecule, or one in a different state ofligation (M. G. Rossmann, ed. “The Molecular Replacement Methods,” Int.Sci. Rev. J. No. 13, Gordon & Breach, New York, N.Y. (1972); EatonLattman, “Use of Rotation and Translation Functions,” H. W. Wyckoff C.H. W. Hist. (S. N. Timasheff, ed.) Methods in Enzymology, 115: 55-77(1985)). For an example of the application of molecular replacement,see, for example, Rice, P. A. & Steitz, T. A. (1994) EMBO J. 13:1514-24.

[0262] In molecular replacement, the three-dimensional structure of theknown molecule is positioned within the unit cell of the new crystal byfinding the orientation and position that provides the best agreementbetween observed diffraction amplitudes and those calculated from theco-ordinates of the positioned subunit. From this modeling, approximatephases for the unknown crystal can be derived. In order to position aknown structure in the unit cells of an unknown, but related structure,three rotation angles and three translations relative to the unit cellorigin have to be determined. The rotation search is carried out bylooking for agreement between the Patterson function of the search andtarget structures as a function of their relative orientation (therotation function). X-PLOR (Brünger et al. (1987) Science 235:458-460;CNS (Crystallography & NMR System, Brünger et al., (1998) Acta Cryst.Sect. D 54: 905-921), and AMORE: an Automatic Package for MolecularReplacement (Navaza, J. (1994) Acta Cryst. Sect. A, 50: 157-163) arecomputer programs that can execute rotation and translation functionsearches. Once the orientation of a test molecule is known, the positionof the molecule must be found using a translational search. Once theknown structure has been positioned in the unit cell of the unknownmolecules, phases for the observed diffraction data can be calculatedfrom the atomic co-ordinates of the structurally related atoms of theknown molecules. By using the calculated phases and X-ray diffractiondata for the unknown molecule, the skilled artisan can generate anelectron density map and/or atomic co-ordinates of the molecule ofinterest.

[0263] By way of example, it is contemplated that a three-dimensionalmodel of a ribosomal subunit other than that derived from H. marismortuican be generated via molecular replacement. In this method, the H.marismortui subunit structures are positioned within the unit cell ofthe new crystal by finding the orientation and position that providesthe best agreement between observed diffraction amplitudes and thosecalculated from the co-ordinates of the positioned subunit. A startingelectron density map calculated using2F_(hk1)(observed)—F_(hk1)(calculated), where F(observed) are thediffraction amplitudes that have been measured from crystals of theunknown structure, and F(calculated) are the diffraction amplitudescalculated from the positioned H. marismortui subunit structure.Refinement of the initial model can be done as is standard in the fieldof macromolecular crystallography.

[0264] The H. marismortui 50S structure can also be used to establishthe structure of a 70S ribosome or 50S ribosome for which an electiondensity map has been calculated, at a resolution that would otherwise betoo low to be interpreted, while a 5 Å resolution map could not beinterpreted in atomic terms de novo, a plausible atomic model can beconstructed by refitting the H. marismortui 50S structure to a lowerresolution map (e.g., 4.5 Å to 8 Å). This refitting can be combined withhomology modeling to obtain a three-dimensional model of a ribosome orribosomal subunit from a different species. It is contemplated thatsimilar procedures may be used to determine the structure of theeukaryotic 60S subunit and/or a eukaryotic ribosome.

[0265] In general, the success of molecular replacement for solvingstructures depends on the fraction of the structures that are relatedand their degree of identity. For example, if about 50% or more of thestructure shows an r.m.s. difference between corresponding atoms in therange of about 2 Å or less, the known structure can be successfully usedto solve the unknown structure.

[0266] Homology modeling, also known as comparative modeling orknowledge-based modeling, can be used to generate a three-dimensionalmodel for a molecule based on the known structure of homologues. Ingeneral, the procedure may comprise one or more of the following steps:aligning the amino acid or nucleic acid sequence of an unknown moleculeagainst the amino acid or nucleic acid sequence of a molecule whosestructure has previously been determined; identifying structurallyconserved and structurally variable regions; generating atomicco-ordinates for core (structurally conserved) residues of the unknownstructure from those of the known structure(s); generating conformationsfor the other (structurally variable) residues in the unknown structure;building side chain conformations; and refining and/or evaluating theunknown structure.

[0267] By way of example, since the nucleotide sequences of all known50S subunit rRNAs can be aligned relative to each other and to H.marismortui 23S and 5S rRNAs, it is possible to construct models of thestructures of other 50S ribosomal rRNAs, particularly in the regions ofthe tunnel and active sites, using the H. marismortui structure.Likewise, homologous proteins can also be modeled using similarmethodologies. Methods useful for comparative RNA sequence analysis areknown in the art and include visual methods and number pattern methods,as well as methods employing chi-square statistics, phylogeneticalgorithms, or empirical algorithms. Descriptions of some of theforegoing methods are available, for example, athttp://www.rna.icmb.utexas.edu/; Gutell (1996), “Comparative SequenceAnalysis and the Structure of 16S and 23S rRNA,” Ribosomal RNA.Structure, Evolution, Processing, and Function in Protein Biosynthesis,(Dahlberg A. and Zimmerman B., eds.) CRC Press. Boca Raton, pp. 111-128;Guttell et al. (1993) Nucl. Acid Res. 21: 3055-3074; Schnare et al.(1996) J. Mol. Biol. 256: 701-719. Particularly useful visual inspectionmethods include comparison of a particular position in a H. marismortuisecondary structure diagram with the residues located at the analogousposition on an E. coli secondary structure diagram. A software programthat is particularly useful in homology modeling includes XALIGN(Wishart, D. et al., (1994) Cabios 10: 687-88). See also, U.S. Pat. No.5,884,230.

[0268] To model the rRNA of a new species, bases of the H. marismortuirRNA can be replaced, using a computer graphics program such as “O”(Jones et al., (1991) Acta Cryst. Sect. A, 47: 110-119), by those of thehomologous rRNA, where they differ. In many if not most cases the sameorientation of the base will be appropriate. Insertions and deletionsmay be more difficult and speculative, but the rRNA forming the peptidyltransferase site and the portion of the tunnel closest to it is veryhighly conserved with essentially no insertions and deletions. Automatedweb-based homology modeling can be performed using, for example, thecomputer programs SWISS-MODEL available through Glaxo WellcomeExperimental Research in Geneva, Switzerland, and WHATIF available onEMBL servers.

[0269] For other descriptions of homology modeling, see, for example,Gutell R. R. (1996), supra; Gutell R. R., et al. (1993) Nucleic AcidsRes. 21: 3055-3074; Schnare et al. (1996) J. Mol. Biol., 256: 701-719;Blundell et al. (1987) Nature 326: 347-352; Fetrow and Bryant (1993)Bio/Technology 11:479-484; Greer (1991) Methods in Enzymology 202:239-252; and Johnson et al. (1994) Crit. Rev. Biochem. Mol. Biol.29:1-68. An example of homology modeling can be found, for example, inSzklarz G. D (1997) Life Sci. 61: 2507-2520.

[0270] As discussed earlier, the large ribosomal subunit fromprokaryotes and eukaryotes are structurally conserved. The amino acidsequences of the large ribosomal subunit from prokaryotes and eukaryotescan be aligned due to the evolutionary conservation of amino acidresidues that are important for three-dimensional structure, the natureand shape of the binding sites for substrates and the catalytic site.This similarity in amino acid sequence of the homologous large ribosomalsubunit allows the construction of models, via homology modeling, forthe molecules whose crystal structures have not been solved.

[0271] The new ribosome or large ribosomal subunit structures determinedusing the H. marismortui crystals and/or atomic co-ordinates can then beused for structure-based drug design using one or more of the approachesdescribed hereinbelow. This information can then be used to designmolecules that selectively bind and disrupt protein synthesis in theribosomes of the pathogens while leaving the ribosomes of a hostrelatively unaffected.

[0272] G. Rational Drug Design

[0273] 1. Introduction

[0274] It is contemplated that the atomic co-ordinates defining a largeribosomal subunit of interest, whether derived from one or more of X-raycrystallography, molecular modeling, homology modeling or molecularreplacement, may be used in rational drug design (RDD) to design a novelmolecule of interest, for example, novel modulators (for example,inducers, mimetics or inhibitors) of ribosome function. Furthermore, itis contemplated that, by using the principles disclosed herein, theskilled artisan can design, make, test, refine and use novel proteinsynthesis inhibitors specifically engineered to reduce, disrupt, orotherwise or inhibit ribosomal function in an organism or species ofinterest. For example, by using the principles discussed herein, theskilled artisan can engineer new molecules that specifically target andinhibit ribosomal function in a pathogen, for example, a particularprokaryotic, organism, while preserving ribosomal function in a host,for example, a eukaryotic organism, specifically a mammal, and morespecifically, a human. As a result, the atomic co-ordinates provided anddiscussed herein permit the skilled artisan to design new antibioticsthat can kill certain pathogenic organisms while having little or notoxicity in the intended recipient, for example, a human.

[0275] It is contemplated that RDD using atomic co-ordinates of thelarge ribosomal subunit can be facilitated most readily viacomputer-assisted drug design (CADD) using conventional computerhardware and software known and used in the art. The candidate moleculesmay be designed de novo or may be designed as a modified version of analready existing molecule, for example, a pre-existing antibiotic, usingconventional methodologies. Once designed, candidate molecules can besynthesized using standard methodologies known and used in the art.Following synthesis, the candidate molecules can be screened forbioactivity, for example, by their ability to reduce or inhibit ribosomefunction, their ability to interact with or bind a ribosome or aribosomal subunit. Based in part upon these results, the candidatemolecules may be refined iteratively using one or more of the foregoingsteps to produce a more desirable molecule with a desired biologicalactivity. The resulting molecules can be useful in treating, inhibitingor preventing the biological activities of target organisms, therebykilling the organism or impeding its growth. Alternatively, theresulting molecules can be useful for treating, inhibiting or preventingmicrobial infections in any organism, particularly animals, moreparticularly humans.

[0276] In summary, the tools and methodologies provided by the presentinvention may be used to identify and/or design molecules which bindand/or interact in desirable ways with ribosomes and ribosomal subunits.Basically, the procedures utilize an iterative process whereby themolecules are synthesized, tested and characterized. New molecules canbe designed based on the information gained in the testing andcharacterization of the initial molecules and then such newly identifiedmolecules can themselves be tested and characterized. This series ofprocesses may be repeated as many times as necessary to obtain moleculeswith desirable binding properties and/or biological activities. Methodsfor identifying candidate molecules are discussed in more detail below.

[0277] 2. Identification of Candidate Molecules

[0278] It is contemplated that the design of candidate molecules ofinterest can be facilitated by conventional ball and stick-type modelingprocedures. However, in view of the size and complexity of the largeribosomal subunit, it is contemplated that the ability to designcandidate molecules may be enhanced significantly using computer-basedmodeling and design protocols.

[0279] a. Molecular Modeling.

[0280] It is contemplated that the design of candidate molecules, asdiscussed in detail hereinbelow, can be facilitated using conventionalcomputers or workstations, available commercially from, for example,Silicon Graphics Inc. and Sun Microsystems, running, for example, UNIXbased, Windows NT on IBM OS/2 operating systems, and capable of runningconventional computer programs for molecular modeling and rational drugdesign.

[0281] It is understood that any computer system having the overallcharacteristics set forth in FIG. 27 may be useful in the practice ofthe invention. More specifically, FIG. 27, is a schematic representationof a typical computer work station having in electrical communication(100) with one another via, for example, an internal bus or externalnetwork, a central processing unit (101), a random access memory (RAM)(102), a read only memory (ROM) (103), a monitor or terminal (104), andoptimally an external storage device, for example, a diskette, CD ROM,or magnetic tape (105).

[0282] The computer-based systems of the invention preferably comprise adata storage means having stored therein a ribosome or ribosomal subunitor fragment sequence and/or atomic co-ordinate/X-ray diffraction data ofthe present invention and the necessary hardware means and softwaremeans for supporting and implementing an analysis means. As used herein,“a computer system” or “a computer-based system” refers to the hardwaremeans, software means, and data storage means used to analyze thesequence, X-ray diffraction data, and/or atomic co-ordinates of theinvention. As used herein, the term “data storage means” is understoodto refer to any memory which can store sequence data, atomicco-ordinates, and/or X-ray diffraction data, or a memory access meanswhich can access manufactures having recorded thereon the atomicco-ordinates of the present invention.

[0283] In one embodiment, a ribosome or ribosomal subunit, or at least asubdomain thereof, amino acid and nucleic acid sequence, X-raydiffraction data and/or atomic co-ordinates of the present invention arerecorded on computer readable medium. As used herein, the term “computerreadable medium” is understood to mean any medium which can be read andaccessed directly by a computer. Such media include, but are not limitedto: magnetic storage media, such as floppy discs, hard disc storagemedium, and magnetic tape; optical storage media such as optical discsor CD-ROM; electrical storage media such as RAM and ROM; and hybrids ofthese categories such as magnetic/optical storage media. A skilledartisan can readily appreciate how any of the presently known computerreadable mediums can be used to create a manufacture comprising computerreadable medium having recorded thereon an amino acid and/or nucleotidesequence, X-ray diffraction data, and/or atomic co-ordinates of thepresent invention.

[0284] As used herein, the term “recorded” is understood to mean anyprocess for storing information on computer readable medium. A skilledartisan can readily adopt any of the presently known methods forrecording information on computer readable medium to generatemanufactures comprising an amino acid or nucleotide sequence, atomicco-ordinates and/or X-ray diffraction data of the present invention.

[0285] A variety of data storage structures are available to a skilledartisan for creating a computer readable medium having recorded thereonamino acid and/or nucleotide sequence, atomic co-ordinates and/or X-raydiffraction data of the present invention. The choice of the datastorage structure will generally be based on the means chosen to accessthe stored information. In addition, a variety of data processorprograms and formats can be used to store the sequence information,X-ray data and/or atomic co-ordinates of the present invention oncomputer readable medium. The foregoing information, data andco-ordinates can be represented in a word processing text file,formatted in commercially-available software such as WordPerfect andMICROSOFT Word, or represented in the form of an ASCII file, stored in adatabase application, such as DB2, Sybase, Oracle, or the like. Askilled artisan can readily adapt any number of data processorstructuring formats (e.g. text file or database) in order to obtaincomputer readable medium having recorded thereon the information of thepresent invention.

[0286] By providing a computer readable medium having stored thereon aribosome or ribosomal subunit sequence, and/or atomic co-ordinates, askilled artisan can routinely access the sequence, and/or atomicco-ordinates to model a ribosome or ribosomal subunit, a subdomainthereof, mimetic, or a ligand thereof. Computer algorithms are publiclyand commercially available which allow a skilled artisan to access thisdata provided in a computer readable medium and analyze it for molecularmodeling and/or RDD. See, e.g., Biotechnology Software Directory,MaryAnn Liebert Publ., New York, N.Y. (1995).

[0287] Although computers are not required, molecular modeling can bemost readily facilitated by using computers to build realistic models ofa ribosome, ribosomal subunit, or a portion thereof. Molecular modelingalso permits the modeling of new smaller molecules, for example ligands,agents and other molecules, that can bind to a ribosome, ribosomalsubunit, or a portion therein. The methods utilized in molecularmodeling range from molecular graphics (i.e., three-dimensionalrepresentations) to computational chemistry (i.e., calculations of thephysical and chemical properties) to make predictions about the bindingof the smaller molecules or their activities; to design new molecules;and to predict novel molecules, including ligands such as drugs, forchemical synthesis.

[0288] For basic information on molecular modeling, see, for example, M.Schlecht, Molecular Modeling on the PC (1998) John Wiley & Sons; Gans etal., Fundamental Principals of Molecular Modeling (1996) Plenum Pub.Corp.; N. C. Cohen, ed., Guidebook on Molecular Modeling in Drug Design(1996) Academic Press; and W. B. Smith, Introduction to TheoreticalOrganic Chemistry and Molecular Modeling (1996). U.S. Patents whichprovide detailed information on molecular modeling include, for example:U.S. Pat. Nos. 6,093,573; 6,080,576; 6,075,014; 6,075,123; 6,071,700;5,994,503; 5,884,230; 5,612,894; 5,583,973; 5,030,103; 4,906,122; and4,812,12.

[0289] Three-dimensional modeling can include, but is not limited to,making three-dimensional representations of structures, drawing picturesof structures, building physical models of structures, and determiningthe structures of related ribosomes, ribosomal subunits andribosome/ligand and ribosomal subunit/ligand complexes using the knownco-ordinates. The appropriate co-ordinates are entered into one or morecomputer programs for molecular modeling, as known in the art. By way ofillustration, a list of computer programs useful for viewing ormanipulating three-dimensional structures include: Midas (University ofCalifornia, San Francisco); MidasPlus (University of California, SanFrancisco); MOIL (University of Illinois); Yummie (Yale University);Sybyl (Tripos, Inc.); Insight/Discover (Biosym Technologies); MacroModel(Columbia University); Quanta (Molecular Simulations, Inc.); Cerius(Molecular Simulations, Inc.); Alchemy (Tripos, Inc.); LabVision(Tripos, Inc.); Rasmol (Glaxo Research and Development); Ribbon(University of Alabama); NAOMI (Oxford University); Explorer Eyechem(Silicon Graphics, Inc.); Univision (Cray Research); Molscript (UppsalaUniversity); Chem-3D (Cambridge Scientific); Chain (Baylor College ofMedicine); O (Uppsala University); GRASP (Columbia University); X-Plor(Molecular Simulations, Inc.; Yale University); Spartan (Wavefunction,Inc.); Catalyst (Molecular Simulations, Inc.); Molcadd (Tripos, Inc.);VMD (University of Illinois/Beckman Institute); Sculpt (InteractiveSimulations, Inc.); Procheck (Brookhaven National Library); DGEOM(QCPE); RE_VIEW (Brunell University); Modeller (Birbeck College,University of London); Xmol (Minnesota Supercomputing Center); ProteinExpert (Cambridge Scientific); HyperChem (Hypercube); MD Display(University of Washington); PKB (National Center for BiotechnologyInformation, NIH); ChemX (Chemical Design, Ltd.); Cameleon (OxfordMolecular, Inc.); and Iditis (Oxford Molecular, Inc.).

[0290] One approach to RDD is to search for known molecular structuresthat might bind to a site of interest. Using molecular modeling, RDDprograms can look at a range of different molecular structures ofmolecules that may fit into a site of interest, and by moving them onthe computer screen or via computation it can be decided whichstructures actually fit the site well (William Bains (1998)Biotechnology from A to Z, second edition, Oxford University Press, p.259).

[0291] An alternative but related approach starts with the knownstructure of a complex with a small molecule ligand and modelsmodifications of that small molecule in an effort to make additionalfavorable interactions with a ribosome or ribosomal subunit.

[0292] The present invention permits the use of molecular and computermodeling techniques to design and select novel molecules, such asantibiotics or other therapeutic agents, that interact with ribosomesand ribosomal subunits. Such antibiotics and other types of therapeuticagents include, but are not limited to, antifiungals, antivirals,antibacterials, insecticides, herbicides, miticides, rodentcides, etc.

[0293] In order to facilitate molecular modeling and/or RDD the skilledartisan may use some or all of the atomic co-ordinates deposited at theRCSB Protein Data Bank with the accession numbers PDB ID: 1FFK, 1JJ2,1FFZ, or 1FG0, and/or those atomic co-ordinates contained on Disk No. 1,2 or 3 of 3. Furthermore, the skilled artisan, using the foregoingatomic co-ordinates, the skilled artisan can generate additional atomicco-ordinates via, for example, molecular modeling using, for example,homology modeling and/or molecular replacement techniques, that togetherdefine at least a portion of a model of a ribosome from another speciesof interest. By using the foregoing atomic co-ordinates, the skilledartisan can design inhibitors of protein synthesis that may be tailoredto be effective against ribosomes from one or more species but whichhave little or no effect on ribosomes of other species. Such inhibitorsmay be competitive inhibitors. As used herein, the term “competitiveinhibitor” refers to an inhibitor that binds to the active form of aribosome or ribosomal subunit at the same sites as its substrate(s) ortRNA(s), thus directly competing with them. The term “active form” of aribosome or ribosomal subunit refers to a ribosome or ribosomal subunitin a state that renders it capable of protein synthesis. Competitiveinhibition can be reversed completely by increasing the substrate ortRNA concentration.

[0294] This invention also permits the design of molecules that act asuncompetitive inhibitors of protein synthesis. As used herein, the term“uncompetitive inhibitor” refers to a molecule that inhibits thefunctional activity of a ribosome or ribosomal subunit by binding to adifferent site on the ribosome or ribosomal subunit than does itssubstrates, or tRNA. Such inhibitors can often bind to the ribosome orribosomal subunit with the substrate or tRNA and not to the ribosome orribosomal subunit by itself. Uncompetitive inhibition cannot be reversedcompletely by increasing the substrate concentration. These inhibitorsmay bind to, all or a portion of, the active sites or other regions ofthe large ribosomal subunit already bound to its substrate and may bemore potent and less non-specific than known competitive inhibitors thatcompete for large ribosomal subunit active sites or for binding to largeribosomal subunit.

[0295] Similarly, non-competitive inhibitors that bind to and inhibitprotein synthesis whether or not it is bound to another chemical entitymay be designed using the atomic co-ordinates of the large ribosomalsubunits or complexes comprising large ribosomal subunit of thisinvention. As used herein, the term “non-competitive inhibitor” refersto an inhibitor that can bind to either the free or substrate or tRNAbound form of the ribosome or ribosomal subunit.

[0296] Those of skill in the art may identify inhibitors as competitive,uncompetitive, or non-competitive by computer fitting enzyme kineticdata using standard equation according to Segel, I. H., (1975) EnzymeKinetics: Behaviour and Analysis of Rapid Equilibrium and Steady-StateEnzyme Systems, (Wiley Classics Library). It should also be understoodthat uncompetitive or non-competitive inhibitors according to thepresent invention may bind the same or different binding sites.

[0297] Alternatively, the atomic co-ordinates provided by the presentinvention are useful in designing improved analogues of known proteinsynthesis inhibitors or to design novel classes of inhibitors based onthe atomic structures and co-ordinates of the crystals of the 50Sribosomal subunit/CCdA-p-Puro complex and the 50S ribosomalsubunit/aa-tRNA analogue complex. This provides a novel route fordesigning inhibitors of protein synthesis with both high specificity,stability and other drug-like qualities (Lipinski et al. (1997) Adv.Drug Deliv. Rev. 23:3).

[0298] The atomic co-ordinates of the present invention also permitprobing the three-dimensional structure of a ribosome or ribosomesubunit or a portion thereof with molecules composed of a variety ofdifferent chemical features to determine optimal sites for interactionbetween candidate inhibitors and/or activators and the ribosome orribosomal subunit. For example, high resolution atomic co-ordinatesbased on X-ray diffraction data collected from crystals saturated withsolvent allows the determination of where each type of solvent moleculesticks. Small molecules that bind to those sites can then be designedand synthesized and tested for their inhibitory activity (Travis, J.(1993) Science 262: 1374). Further, any known antibiotic, inhibitor orother small molecule that binds to the H. marismortui large subunit canbe soaked into H. marismortui large subunit crystals and their exactmode of binding determined from difference electron density maps. Thesemolecules may represent lead compounds from which better drug-likecompounds can be synthesized.

[0299] b. Identification of Target Sites.

[0300] The atomic co-ordinates of the invention permit the skilledartisan to identify target locations in a ribosome or large ribosomalsubunit that can serve as a starting point in rational drug design. As athreshold matter, the atomic co-ordinates of the invention permit theskilled artisan to identify specific regions within a ribosome orribosomal subunit that are involved with protein synthesis and/orprotein secretion out of the ribosome. Furthermore, the atomicco-ordinates of the invention permit a skilled artisan to furtheridentify portions of these regions that are conserved or are notconserved between different organisms. For example, by identifyingportions of these regions that are conserved among certain pathogens,for example, certain prokaryotes, but are not conserved in a hostorganism, for example, a eukaryote, more preferably a mammal, theskilled artisan can design molecules that selectively inhibit or disruptprotein synthesis activity of the pathogen's but not the host'sribosomes. Furthermore, by analyzing regions that are either conservedor non-conserved between certain pathogens, it may be possible to designbroad or narrow spectrum protein synthesis inhibitors, e.g.,antibiotics, as a particular necessity arises.

[0301]FIG. 28, is a schematic representation of a large ribosomalsubunit that identifies a variety of exemplary target sites that appearto participate in protein synthesis within the ribosome and/or theexport or translocation of the newly synthesized protein out of theribosome. The target sites include, for example, the P-site (200), theA-site (201), the peptidyl transferase center (202), the peptidyltransferase site (203) which includes at least a portion of the P-siteand the A-site, a factor binding domain (204) including, for example,the EF-Tu binding domain and the EF-G binding domain, the polypeptideexit tunnel (205) including cavities defined by the wall of the exittunnel, and the signal recognition particle binding domain (206).

[0302] By way of example, inspection of the atomic co-ordinates of theH. marismortui 50S ribosomal subunit has identified a variety of targetregions that may serve as a basis for the rational drug design of new ormodified protein synthesis inhibitors. The target regions include thepeptidyl transferase site, A-site, the P-site, the polypeptide exittunnel, certain cavities disposed in the wall of the polypeptide exittunnel (for example, cavity 1 and cavity 2), and certain antibioticbinding pockets. The residues that together define at least a portion ofeach of the foregoing regions are identified in the following tables.However, it is contemplated that the same or similar target sites can beidentified in a ribosome or a ribosomal unit of interest using theprinciples described herein. Furthermore, these principles can beemployed using any of the primary sets of atomic co-ordinates providedherein or any additional atomic co-ordinate sets, for example, secondaryatomic co-ordinate sets that may be generated by molecular modeling ofany ribosome or ribosomal subunit of interest.

[0303] Table 5 identifies the residues in the H. marismortui 50Sribosomal subunit that together define at least a portion of theribosomal peptidyl transferase site. In addition, Table 5 identifieswhich of those residues that define at least a portion of the peptidyltransferase site are not conserved between H. marismortui and E. coli,those that are not conserved between H. marismortui and rat, those thatare not conserved between E. coli and rat, and those that are notconserved between eubacteria and eukaryota. The non-conserved residueswere identified by comparison of sequences of H. marismortui 23S rRNAthat form the above-mentioned sites with the corresponding sequences ofaligned rRNA from the other organisms. TABLE 5 Residues that Define theRibosomal Peptidyl-Transferase Site Residues Not Residues Not ResiduesNot Conserved between Conserved Between Conserved Between ResidueConserved H. marismortui H. marismortui and H. marismortui and E. coliand Rattus Between Bacteria A-site Residues E. coli Rattus (Coli/Rat)Eukaryotes? G2102 Yes A2103 Yes C2104 Yes C2105 Yes C2106 Yes G2482 YesG2284 Yes G2285 Yes G2286 Yes A2474 Yes A2485 Yes A2486 Yes C2487 YesA2488 U A/U No U2528 Yes C2536 Yes A2538 Yes G2540 Yes U2541 Yes C2542Yes G2543 Yes G2588 Yes U2589 Yes U2590 Yes C2608 Yes G2617 Yes G2618Yes U2619 Yes U2620 Yes A2635 Yes C2636 Yes A2637 Yes G2638 Yes

[0304] Table 6 identifies the residues in the H. marismortui 50Sribosomal subunit that together define at least a portion of theribosomal A-site. In addition, Table 6 identifies which of thoseresidues that define at least a portion the A-site are not conservedbetween H. marismortui and E. coli, those that are not conserved betweenH. marismortui and rat, those that are not conserved between E. coli andrat, and those that are not conserved between eubacteria and eukaryota.The non conserved residues were identified as described previously withrespect to Table 5. TABLE 6 Residues that Define the Ribosomal A-siteResidues Not Residues Not Residues Not Conserved between ConservedBetween Conserved Between Residue Conserved H. marismortui H.marismortui and H. marismortui and E. coli and Rattus Betweeen Bacteriaand A-site Residues E. coli Rattus (Coli/Rat) Eukaryotes? G2102 YesA2103 Yes C2104 Yes G2482 Yes A2485 Yes A2486 Yes C2487 Yes A2488 U A/UNo U2528 Yes C2536 Yes A2538 Yes G2540 Yes U2541 Yes C2542 Yes G2543 YesG2588 Yes U2589 Yes U2590 Yes C2608 Yes G2617 Yes G2618 Yes U2619 YesU2620 Yes A2637 Yes

[0305] Table 7 identifies the residues in the H. marismortui 50Sribosomal subunit that together define at least a portion of theribosomal P-site. As demonstrated in Table 7, all of the residues inthat portion of the ribosomal P-sites are conserved between H.marismortui and E. coli, between H. marismortui and rat, between E. coliand rat, and between eubacteria and eukaryota, as determined using thecomparison method described previously with respect to Table 5. TABLE 7Residues that Define the Ribosomal P-site Residues Not Residues NotResidues Not Conserved between Conserved Between Conserved BetweenResidue Conserved H. marismortui H. marismortui and H. marismortui andE. coli and Rattus Between Bacteria and P-site Residues E. coli Rattus(Coli/Rat) Eukaryotes? C2104 Yes C2105 Yes C2106 Yes G2284 Yes G2285 YesG2286 Yes A2474 Yes A2485 Yes A2486 Yes U2619 Yes U2620 Yes A2635 YesC2636 Yes A2637 Yes G2638 Yes

[0306] Table 8 identifies the residues in the H. marismortui 50Sribosomal subunit that together define at least a portion of theribosomal polypeptide exit tunnel. In addition, Table 8 identifies whichof those residues that define at least a portion of the polypeptide exittunnel are not conserved between H. marismortui and E. coli, those thatare not conserved between H. marismortui and rat, those that are notconserved between E. coli and rat, and those that are not conservedbetween eubacteria and eukaryota. The non conserved residues wereidentified as described previously with respect to Table 5. TABLE 8Residues that Define the Ribosomal Peptide Exit Tunnel Residues NotResidues Not Residues Not Conserved between Conserved Between ConservedBetween Residue Conserved H. marismortui H. marismortui and H.marismortui and E. coli and Rattus Between Bacteria and A-site ResiduesE. coli Rattus (Coli/Rat) Eukaryotes? 23S rRNA G23 Yes G24 Yes A60 YesG88 Yes G89 Yes A90 Yes U454 Yes A462 C C/A No A466 Yes G467 C A C/A NoU468 C C Yes G469 Yes A476 Yes A477 Yes C478 A A Yes G487 Yes U488 A AYes A489 Yes C490 Yes C491 U C/U No C492 U C/U No A497 Yes A498 G A/G NoG499 A G/A No G500 G501 A G/A No A513 G A/G No G514 A A/G No G636 A G/ANo A767 Yes U768 A A Yes U835 del del/U No C839 U G U/G No U840 Yes A841G G Yes A844 Yes U845 A A Yes C879 Yes A882 Yes U883 Yes C884 Yes G885 AC A/C No U888 C C Yes C889 Yes C890 G A G/A No U1359 Yes C1360 G G/C NoC1361 U C/U No U1362 Yes G1363 Yes G1364 A A/G No C1365 U C/U No C1366 GA/G No A1367 U G U/G No A1424 U G U/G No G1425 C U C/U No C1426 G C/G NoA1427 Yes C1428 A A Yes U1429 C C Yes G1430 A G/A No C1439 G A G/A NoU1440 G C G/C No G1441 C A C/A No A1442 U A/U No A1689 C A/C No C1690 AC/A No A1691 G A/G No A1836 Yes G1837 U A U/A No U1838 Yes A2054 YesA2055 Yes C2056 A A Yes U2057 C U/C No G2058 U C U/C No C2098 G A G/A NoG2099 A A/G No A2100 Yes A2101 Yes G2102 Yes A2103 Yes C2104 Yes C2105Yes A2474 Yes C2475 Yes C2476 U U/C No C2477 A C/A No U2478 C C YesG2482 Yes A2485 Yes A2486 Yes C2487 Yes A2488 U A/U No C2536 Yes A2538Yes G2540 Yes U2541 Yes C2542 Yes C2608 Yes G2611 Yes G2616 Yes G2618Yes U2619 Yes U2620 Yes U2621 Yes A2637 Yes G2643 Yes C2644 U U YesU2645 C C/U No C2646 C U C/U No C2647 U C/U No Protein L4 E59 Yes S60 VS V/S No F61 T W T/W No G62 Yes S63 T S/T No G64 Yes R65 DEL A DEL/A NoQ67 DEL V DEl/V No A68 DEL DEL/A No H69 K R K/R No V70 P/ I P/I No P71 WP W/P No K72 R R Yes L73 K R K/R No D74 G G Yes G75 T G T/G No R76 G GYes A77 R T R/T No Protein L22 E20 H N H/N No E121 S K S/K No Q122 M MYes Q123 K R K/R No G124 R R Yes R125 I R I/R No

[0307]FIG. 26 shows a region of the large ribosomal subunit in which anantibiotic binds. FIG. 26(A) shows an enlarged portion of the largeribosomal subunit with the antibiotic tylosin bound at the top of thepolypeptide exit tunnel adjacent the peptidyl transferase site. FIGS.26(B) and 26(C) are views showing each half of a large ribosomal subunitcut along the polypeptide exit tunnel and are provided to orient thereader to show the tylosin binding site relative to the large ribosomalunit as a whole. FIG. 26(A) also shows two cavities defined by the wallof the polypeptide exit tunnel and are denoted as “cavity 1” and “cavity2.” In addition, FIG. 26(A) also shows a disaccharide binding pocket.The direction in which the newly synthesized polypeptide chains exitsthe ribosome through the polypeptide exit tunnel is denoted by an arrow.

[0308] Table 9 identifies the residues in the H. marismortui 50Sribosomal subunit that together define a first cavity within the wall ofpolypeptide exit tunnel (cavity 1). In addition, Table 9 identifieswhich of those residues that define cavity 1 are not conserved betweenH. marismortui and E. coli, those that are not conserved between H.marismortui and rat, those that are not conserved between E. coli andrat, and those that are not conserved between eubacteria and eukaryota.The non-conserved residues were identified as described previously withrespect to Table 5. TABLE 9 Residues that Define Cavity 1 in theRibosomal Peptide Exit Tunnel Residues Not Residues Not Residues NotConserved between Conserved Between Conserved Between Residue ConservedH. marismortni H. marismortni and H. marismortni and E. coli and RattusBetween Bacteria and Residues E. coli Rattus (Coli/Rat) Eukaryotes? C474G G Yes A766 Yes A767 Yes U768 A C A/C No U883 Yes C884 Yes G885 A A/GNo A886 Yes U888 C C Yes C889 Yes C890 G A G/A No U1359 Yes G1837 U AU/A No A2100 Yes A2101 Yes G2102 Yes A2103 Yes C2475 Yes C2476 U U/C NoC2477 A C/A No U2478 C C Yes A2479 G G/A No A2538 Yes Protein L4 P57 R SR/S No A58 Yes E59 Yes S60 V V/S No F61 T W T/W No G62 Yes S63 T S/T NoG64 Yes R65 K K/R No Q67 DEL V DEL/V No V70 P I P/I No P71 W W/P No K72R R Yes L73 K R K/R No D74 G G Yes G75 T T/G No R76 G G Yes

[0309] Table 10 identifies the residues in the H. marismortui 50Sribosomal subunit that together define a second cavity in the wall ofpolypeptide exit tunnel (cavity 2). In addition, Table 10 identifieswhich of those residues that define cavity 2 are not conserved betweenH. marismortui and E. coli, those that are not conserved between H.marismortui and rat, those that are not conserved between E. coli andrat, and those that are not conserved between eubacteria and eukaryota.The non conserved residues were identified as described previously withrespect to Table 5. TABLE 10 Residues that Define Cavity 2 in theRibosomal Peptide Exit Tunnel Residues Not Residues Not Residues NotConserved between Conserved Between Conserved Between Residue ConservedH. marismortui H. marismortui and H. marismortui and E. coli and RattusBetween Bacteria and Residues E. coli Rattus (Coli/Rat) Eukaryotes? U831C G C/G No U832 DEL DEL/U No G833 U C U/C No G834 A C A/C No U835 DELDEL/U No G836 A A/G No U837 A U/A No C838 G G/C No C839 U G U/G No U840Yes A841 G G Yes A843 Yes A844 Yes U845 A A Yes A846 U A/U No C847 U U/CNo C848 U G U/G No C849 A G A/G No C1753 Yes A1754 Yes G1837 U A U/A NoU1838 Yes A1839 Yes G2099 A A/G No A2100 Yes A2103 Yes U2615 Yes G2616Yes U2621 Yes A2622 Yes G2643 Yes C2644 U U Yes U2645 C C/U No G2646 C UC/U No C2647 U C/U No

[0310] Tables 9 and 10, however, define only two or many cavitiesdisposed within the wall of the polypeptide exit tunnel. However, byusing the atomic co-ordinates and molecular modeling methodologiesdescribed herein, the skilled artisan may identify the residues(contributed by amino acids, nucleotides or a combination of both) thattogether define other cavities within the wall of the polypeptide exittunnel.

[0311] In addition, by using the atomic co-ordinates described herein,the skilled artisan can identify the antibiotic binding site of anyantibiotic of interest. This information also provides contact sitesbetween an antibiotic and the residues in a ribosome or ribosomalsubunit, which can be used to advantage in the design of novel ormodified protein synthesis inhibitors. The binding or contact sites fora variety of antibiotics are discussed in more detail below.

[0312] Table 11 identifies the residues in the H. marismortui 50Sribosomal subunit that together define at least a portion of ananisomycin binding pocket. In addition, Table 11 identifies which ofthose residues that define at least a portion of the anisomycin bindingpocket are not conserved between H. marismortui and E. coli, those thatare not conserved between H. marismortui and rat, those that are notconserved between E. coli and rat, and those that are not conservedbetween eubacteria and eukaryota. The non-conserved residues wereidentified as described previously with respect to Table 5. TABLE 11Residues that Define the Anisomycin Binding Pocket Residues Not ResiduesNot Residues Not Conserved between Conserved Between Conserved BetweenResidue Conserved H. marismortui H. marismortui and H. marismortui and Ecoli. and Rattus Between Bacteria and Residues E. coli Rattus (Coli/Rat)Eukaryotes? G2102 Yes G2482 Yes A2486 Yes C2487 Yes A2488 U A/U No U2535Yes A2538 Yes U2539 Yes G2540 Yes U2541 Yes

[0313] Table 12 identifies the residues in the H. marismortui 50Sribosomal subunit that together define at least a portion of ablasticidin binding pocket. As demonstrated in Table 12, all of theresidues in that portion of the blasticidin binding pocket are conservedbetween H. marismortui and E. coli, between H. marismortui and rat,between E. coli and rat, and between eubacteria and eukaryota, asdetermined using the comparison method described previously with respectto Table 5. TABLE 12 Residues that Define the Blasticidin Binding PocketResidues Not Residues Not Residues Not Conserved between ConservedBetween Conserved Between Residue Conserved H. marismortui H.marismortui and H. marismortui and E. coli and Rattus Between Bacteriaand Residues E. coli Rattus (Coli/Rat) Eukaryotes? C2104 Yes C2105 YesC2106 Yes G2284 Yes G2285 Yes U2473 Yes A2474 Yes A2485 Yes A2486 YesU2620 Yes G2634 Yes A2635 Yes C2636 Yes A2637 Yes

[0314] Table 13 identifies the residues in the H. marismortui 50Sribosomal subunit that together define at least a portion of acarbomycin binding pocket. In addition, Table 13 identifies which ofthose residues that define at least a portion of the carbomycin bindingpocket are not conserved between H. marismortui and E. coli, those thatare not conserved between H. marismortui and rat, those that are notconserved between E. coli and rat, and those that are not conservedbetween eubacteria and eukaryota. The non-conserved residues wereidentified as described previously with respect to Table 5. TABLE 13Residues that Define the Carbomycin Binding Pocket Residues Not ResiduesNot Residues Not Conserved between Conserved Between Conserved BetweenResidue Conserved H. marismortui H. marismortui E. coli and RattusBetween Bacteria H. marismortui Residues and E. coli and Rattus(Coli/Rat) and Eukaryotes? C839 U G U/G No G2099 A A/G No A2100 YesG2102 Yes A2103 Yes C2104 Yes A2486 Yes C2487 Yes A2538 Yes G2540 YesU2541 Yes U2620 Yes C2644 U U Yes G2646 C U C/U No

[0315] Table 14 identifies the residues in the H. marismortui 50Sribosomal subunit that together define at least a portion of a tylosinbinding pocket. In addition, Table 14 identifies which of those residuesthat define at least a portion of the tylosin binding pocket are notconserved between H. marismortui and E. coli, those that are notconserved between H. marismortui and rat, those that are not conservedbetween E. coli and rat, and those that are not conserved betweeneubacteria and eukaryota. The non-conserved residues were identified asdescribed previously with respect to Table 5. TABLE 14 Residues thatDefine the Tylosin Binding Pocket Residues Not Residues Not Residues NotConserved between Conserved Between Conserved Between Residue ConservedH. marismortui H. marismortui E. coli and Rattus Between Bacteria H.marismortui Residues and E. coli and Rattus (Coli/Rat) and Eukaryotes?C839 U G U/G No A841 G G Yes A843 Yes A844 Yes U845 A A Yes G1837 U AU/A No C2098 G A G/A No G2099 A A/G No A2100 Yes G2102 Yes A2103 YesA2538 Yes U2539 Yes G2540 Yes U2541 Yes U2645 C C/U No G2646 C U C/U No

[0316] Table 15 identifies the residues in the H. marismortui 50Sribosomal subunit that together define at least a portion of asparsomycin binding pocket. As demonstrated in Table 15, all of theresidues in that portion of the sparsomycin binding pocket are conservedbetween H. marismortui and E. coli, between H. marismortui and rat,between E. coli and rat, and between eubacteria and eukaryota, asdetermined using the comparison method described previously with respectto Table 5. TABLE 15 Residues that Define the Sparsomycin Binding PocketResidues Not Residues Not Residues Not Conserved between ConservedBetween Conserved Between Residue Conserved H. marismortui H.marismortui E. coli and Rattus Between Bacteria H. marismortui Residuesand E. coli and Rattus (Coli/Rat) and Eukaryotes? A2486 Yes C2487 YesU2541 Yes C2608 Yes U2619 Yes U2620 Yes C2636 Yes A2637 Yes

[0317] Table 16 identifies the residues in the H. marismortui 50Sribosomal subunit that together define at least a portion of avirginiamycin binding pocket. As demonstrated in Table 16, all of theresidues in that portion of the virginiamycin binding pocket areconserved between H. marismortui and E. coli, between H. marismortui andrat, between E. coli and rat, and between eubacteria and eukaryota, asdetermined using the comparison method described previously with respectto Table 5. TABLE 16 Residues that Define the Virginiamycin BindingPocket Residues Not Residues Not Residues Not Conserved betweenConserved Between Conserved Between Residue Conserved H. marismortui H.marismortui E. coli and Rattus Between Bacteria H. marismortui Residuesand E. coli and Rattus (Coli/Rat) and Eukaryotes? A2100 Yes G2102 YesA2103 Yes C2104 Yes C2105 Yes G2482 Yes A2486 Yes C2487 Yes U2535 YesC2536 Yes A2538 Yes U2539 Yes G2540 Yes U2541 Yes U2620 Yes

[0318] Table 17 identifies the residues in the H. marismortui 50Sribosomal subunit that together define at least a portion of aspiramycin binding pocket. In addition, Table 17 identifies which ofthose residues that define at least a portion of the spiramycin bindingpocket are not conserved between H. marismortui and E. coli, those thatare not conserved between H. marismortui and rat, those that are notconserved between E. coli and rat and those that are not conservedbetween eubacteria and eukaryota. The non-conserved residues wereidentified as described previously with respect to Table 5. TABLE 17Residues that Define the Spiramycin Binding Pocket Residues Not ResiduesNot Residues Not Conserved between Conserved Between Conserved BetweenResidue Conserved H. marismortui H. marismortui E. coli and RattusBetween Bacteria H. marismortui Residues and E. coli and Rattus(Coli/Rat) and Eukaryotes? C839 U G U/G No C2098 G A G/A No G2099 A A/GNo A2100 Yes G2102 Yes A2103 Yes A2538 Yes U2539 Yes G2540 Yes U2541 YesC2644 U U Yes G2646 C U C/U No

[0319] The skilled artisan, when in possession of the foregoing or otherexemplary target sites, may use the process of rational drug design toidentify molecules that potentially bind to one or more of the targetsites and/or inhibit ribosomal activity. Furthermore, by taking intoaccount which of the residues that define the target site are conservedbetween pathogens but not conserved between host species, the skilledartisan can design new species-specific protein synthesis inhibitors. Itis apparent that the skilled artisan can take advantage of the regionsthat are not conserved between E. coli and rat to provide target regionsfor rational drug design. By way of example, FIG. 29 shows certainregions of the polypeptide exit tunnel that are conserved between E.coli and rat (denoted in red) and regions of the polypeptide exit tunnelthat are not conserved between E. coli and rat (denoted in blue). FIGS.29(A) and 29(B) provide enlarged views of a large ribosomal subunit whencut in half along the polypeptide exit tunnel. FIG. 29(C) is provided toorient the reader to the view in FIG. 29(A) relative to the largeribosomal subunit. FIG. 29(D) is provided to orient the reader to theview in FIG. 29B relative to the large ribosomal subunit. In addition,the skilled artisan when in possession of mutations that prevent orreduce antibiotic activity (i.e., are related to antibiotic resistance)can use this information to model the relevant antibiotic bindingproduct which can then be used as a basis for rational drug design toidentify small molecules that overcome drug resistance. It iscontemplated that a variety of computer modeling procedures, forexample, homology modeling protocols, can be used to provide a model ofa drug resistance target site by implementing site directed mutagenesisof nucleotides and/or amino acids and then using the appropriate energyminimization and refinement protocols.

[0320] C. Identification of Candidate Molecules.

[0321] It is contemplated that candidate molecules that inhibit proteinbiosynthesis can be designed entirely de novo or may be based upon apre-existing protein biosynthesis inhibitor. Either of these approachescan be facilitated by computationally screening databases and librariesof small molecules for chemical entities, agents, ligands, or compoundsthat can bind in whole, or in part, to ribosomes and ribosomal subunits,more preferably to large ribosomal subunits, and even more preferably to50S ribosomal subunits. In this screening, the quality of fit of suchentities or compounds to the binding site or sites may be judged eitherby shape complementarity or by estimated interaction energy (Meng et al.(1992) J. Coma. Chem. 13: 505-524).

[0322] The design of molecules that bind to or inhibit the functionalactivity of ribosomes or ribosomal subunits according to this inventiongenerally involves consideration of two factors. First, the moleculemust be capable of physically and structurally associating with thelarge ribosomal subunit. Non-covalent molecular interactions importantin the association of ribosomes and ribosomal subunits with themolecule, include hydrogen bonding, van der Waals and hydrophobicinteractions. Second, the molecule must be able to assume a conformationthat allows it to associate with the ribosomes or ribosomal subunits,more preferably with the large ribosomal subunits, and even morepreferably with the 50S ribosomal subunit. Although certain portions ofthe molecule may not directly participate in this association with aribosome or ribosomal subunits those portions may still influence theoverall conformation of the molecule. This, in turn, may have asignificant impact on binding affinities, therapeutic efficacy,drug-like qualities, and potency. Such conformational requirementsinclude the overall three-dimensional structure and orientation of thechemical entity or molecule in relation to all or a portion of theactive site or other region of the ribosomes or ribosomal subunits, orthe spacing between functional groups of a molecule comprising severalchemical entities that directly interact with the ribosomes or ribosomalsubunits, more preferably with the large ribosomal subunits, and evenmore preferably with the 50S ribosomal subunit.

[0323] The potential, predicted, inhibitory or binding effect of amolecule on ribosomes and ribosomal subunits may be analyzed prior toits actual synthesis and testing by the use of computer modelingtechniques. If the theoretical structure of the given molecule suggestsinsufficient interaction and association between it and ribosomes orribosomal subunits, synthesis and testing of the molecule is obviated.However, if computer modeling indicates a strong interaction, themolecule may then be synthesized and tested for its ability to interactwith the ribosomes or ribosomal subunits and inhibit protein synthesis.In this manner, synthesis of inoperative molecules may be avoided. Insome cases, inactive molecules are synthesized predicted on modeling andthen tested to develop a SAR (structure-activity relationship) formolecules interacting with a specific region of the ribosome orribosomal subunit, more preferably of the large ribosomal subunit, andeven more preferably of the 50S ribosomal subunit. As used herein, theterm “SAR”, shall collectively refer to the structure-activity/structureproperty relationships pertaining to the relationship(s) between acompound's activity/properties and its chemical structure.

[0324] d. De Novo Design.

[0325] One skilled in the art may use one of several methods to identifychemical moieties or entities, compounds, or other agents for theirability to associate with a preselected target site within a ribosomesor ribosomal subunit. This process may begin by visual inspection orcomputer assisted modeling of, for example, the target site on thecomputer screen based on the atomic co-ordinates of the 50S ribosomalsubunit and/or its complexes with other analogues and antibiotics,deposited in the RCSB Protein Data Bank with accession numbers PDB ID:1FFK, 1JJ2, 1FFZ, or 1FG0, and/or listed in a table contained on DiskNo. 1, 2 or 3 of 3. In one embodiment, compound design uses computermodeling programs which calculate how different molecules interact withthe various sites of the ribosome, ribosomal subunit, or a fragmentthereof. Selected chemical moieties or entities, compounds, or agentsmay then be positioned in a variety of orientations, or docked, withinat least a portion of the target site of a ribosome or ribosomalsubunit, more preferably of a large ribosomal subunit, and even morepreferably of a 50S ribosomal subunit. Databases of chemical structuresare available from, for example, Cambridge Crystallographic Data Center(Cambridge, U.K.) and Chemical Abstracts Service (Columbus, Ohio).Docking may be accomplished using software such as Quanta and Sybyl,followed by energy minimization and molecular dynamics with standardmolecular mechanics forcefields, such as CHARMM and AMBER.

[0326] Specialized computer programs may also assist in the process ofselecting chemical entities. These include, but are not limited to:

[0327] (1) GRID (Goodford, P. J., “A Computational Procedure forDetermining Energetically Favorable Binding Sites on BiologicallyImportant Macromolecules” (1985) J. Med. Chem. 28, 849-857). Softwaresuch as GRID, a program that determines probable interaction sitesbetween probes with various functional group characteristics and themacromolecular surface, can be used to analyze the surface sites todetermine structures of similar inhibiting proteins or molecules. TheGRID calculations, with suitable inhibiting groups on molecules (e.g.,protonated primary amines) as the probe, are used to identify potentialhotspots around accessible positions at suitable energy contour levels.

[0328] GRID is available from Oxford University, Oxford, UK.

[0329] (2) MCSS (Miranker, A. and M. Karplus (1991) “Functionality Mapsof Binding Sites: A Multiple Copy Simultaneous Search Method.” Proteins:Structure, Function and Genetics 11: 29-34). MCSS is available fromMolecular Simulations, Burlington, Mass.

[0330] (3) AUTODOCK (Goodsell, D. S. and A. J. Olsen (1990) “AutomatedDocking of Substrates to Proteins by Simulated Annealing” Proteins:Structure, Function, and Genetics 8: 195-202). AUTODOCK is availablefrom Scripps Research Institute, La Jolla, Calif.

[0331] (4) DOCK (Kuntz, I. D. et al. (1982) “A Geometric Approach toMacromolecule-Ligand Interactions” J. Mol. Biol. 161: 269-288). Theprogram DOCK may be used to analyze an active site or ligand bindingsite and suggest ligands with complementary steric properties. DOCK isavailable from University of California, San Francisco, Calif.

[0332] (5) ALADDIN (Van Drie et al. (1989) “ALADDIN: An Integrated Toolof Computer Assisted Molecular Design and Pharmacophore Recognition FromGeometric, Steric and Substructure Searching of Three-DimensionalStructures” J. Comp-Aided Mol. Des. 3: 225).

[0333] (6) CLIX (Davie and Lawrence (1992) “CLIX: A Search Algorithm forFunding Novel Ligands Capable of Binding Proteins of KnownThree-Dimensional Structure” Proteins 12: 31-41).

[0334] (7) GROUPBUILD (Rotstein and Murcko (1993) “GroupBuild: AFragment-Based Method for De Novo Drug Design” J. Med. Chem 36: 1700).(8) GROW (Moon and Howe (1991) “Computer Design of Bioactive Molecules:A Method for Receptor-Based De Novo Ligand Design” Proteins 11: 314).

[0335] Once suitable chemical moieties or entities, compounds, or agentshave been selected, they can be assembled into a single molecule.Assembly may proceed by visual inspection and/or computer modeling andcomputational analysis of the spatial relationship of the chemicalmoieties or entities, compounds or agents with respect to one another inthree-dimensional space. This could then be followed by model buildingusing software such as Quanta or Sybyl.

[0336] Useful programs to aid one of skill in the art in connecting theindividual chemical entities, compounds, or agents include but are notlimited to:

[0337] (1) CAVEAT (Bartlett, P. A. et al. (1989) “CAVEAT: A Program toFacilitate the Structure-Derived Design of Biologically ActiveMolecules”. In molecular Recognition in Chemical and BiologicalProblems”, Special Pub., Royal Chem. Soc. 78: 82-196) and (Bacon et al.(1992) J. Mol. Biol. 225: 849-858). CAVEAT uses databases of cycliccompounds which can act as “spacers” to connect any number of chemicalfragments already positioned in the active site. This allows one skilledin the art to quickly generate hundreds of possible ways to connect thefragments already known or suspected to be necessary for tight binding.CAVEAT is available from the University of California, Berkeley, Calif.

[0338] (2) 3D Database systems such as MACCS-3D (MDL InformationSystems, San Leandro, (Calif.). This area is reviewed in Martin, Y. C.,(1992) “3D Database Searching in Drug Design”, J. Med. Chem. 35:2145-2154.

[0339] (3) HOOK (available from Molecular Simulations, Burlington,Mass.).

[0340] Instead of proceeding to build a molecule of interest in astep-wise fashion one chemical entity at a time as described above, themolecule of interest may be designed as a whole using either an emptyactive site or optionally including some portion or portions of a knowninhibitor or inhibitors. Software that implements these methods include:

[0341] (1) LUDI (Bohm, H.-J. (1992) “The Computer Program LUDI: A NewMethod for the De Novo Design of Enzyme Inhibitors”, J. ComR. Aid.Molec. Design 6: 61-78). The program LUDI can determine a list ofinteraction sites into which to place both hydrogen bonding andhydrophobic fragments. LUDI then uses a library of approximately 600linkers to connect up to four different interaction sites intofragments. Then smaller “bridging” groups such as —CH₂— and —COO— areused to connect these fragments. For example, for the enzyme DHFR, theplacements of key functional groups in the well-known inhibitormethotrexate were reproduced by LUDI. See also, Rotstein and Murcko,(1992) J. Med. Chem. 36:1700-1710. LUDI is available from BiosymTechnologies, San Diego, Calif.

[0342] (2) LEGEND (Nishibata, Y. and A. Itai (1991) Tetrahedron 47,8985). LEGEND is available from Molecular Simulations, Burlington, Mass.

[0343] (3) LeapFrog (available from Tripos Associates, St. Louis, Mo.).

[0344] (4) Aladdin (available from Daylight Chemical InformationSystems, Irvine, Calif.)

[0345] Other molecular modeling techniques may also be employed inaccordance with this invention. See, e.g, Cohen, N. C. et al. (1990)“Molecular Modeling Software and Methods for Medicinal Chemistry, J. MedChem. 33: 883-894. See also, Navia, M. A. and M. A. Murcko (1992) “TheUse of Structural Information in Drug Design”, Current Opinions inStructural Biology 2: 202-210; and Jorgensen (1998) “BOSS-Biochemicaland Organic Simulation System” in the Encyclopedia of ComputationalChemistry (P. V. R. Schleyer, ed.) Wiley & Sonstra., Athens, U.S.A. 5:3281-3285).

[0346] It is contemplated that during modeling, it may be possible tointroduce into the molecule of interest, chemical moieties that may bebeneficial for a molecule that is to be administered as apharmaceutical. For example, it may be possible to introduce into oromit from the molecule of interest, chemical moieties that may notdirectly affect binding of the molecule to the target area but whichcontribute, for example, to the overall solubility of the molecule in apharmaceutically acceptable carrier, the bioavailability of the moleculeand/or the toxicity of the molecule. Considerations and methods foroptimizing the pharmacology of the molecules of interest can be found,for example, in “Goodman and Gilman's The Pharmacological Basis ofTherapeutics” Eighth Edition (Goodman Gilman, Rall, Nies, & Taylor(eds.)). Pergaman Press (1985); Jorgensen & Duffy (2000) Bioorg. Med.Chem. Lett. 10: 1155-1158.

[0347] Furthermore, the computer program “Qik Prop” can be used toprovide rapid predictions for physically significant descriptions andpharmaceutically-relevant properties of an organic molecule of interest.A ‘Rule of Five’ probability scheme can be used to estimate oralabsorption of the newly synthesized compounds (Lipinski et al. (1997)Adv. Drug Deliv. Rev. 23:3).

[0348] Programs suitable for pharmacophore selection and design include:

[0349] (1) DISCO (Abbot Laboratories, Abbot Park, Ill.).

[0350] (2) Catalyst (Bio-CAD Corp., Mountain View, Calif.).

[0351] (3) Chem DBS-3D (Chemical Design Ltd., Oxford, U.K.).

[0352] Furthermore, the skilled artisan may use the informationavailable on how to design suitable therapeutically active andpharmaceutically useful compounds, and use this information in thedesign of new protein synthesis inhibitors of the invention. See, forexample, Lipinski et al. (1997) Ad. Drug Deliv. Reviews 23: 3-25; Van deWaterbeemd et al. (1996) Quantitative Structure-Activity Relationships15: 480-490; and Cruciani et al (2000), Theochem-J. Mol. Struct. 503:17-30.

[0353] The entry of the co-ordinates of the ribosome's or ribosomalsubunit's proteins and RNAs into the computer programs discussed aboveresults in the calculation of most probable structure of themacromolecule, including overall atomic co-ordinates of a ribosome,ribosomal subunit or a fragment thereof. These structures can becombined and refined by additional calculations using such programs todetermine the probable or actual three-dimensional structure of theribosome, ribosomal subunit or a fragment thereof, including potentialor actual active or binding sites of ligands.

[0354] e. Modification of Existing Molecules.

[0355] Instead of designing molecules of interest entirely de novo it iscontemplated that pre-existing molecules or proteins thereof may be usedas a starting point for the design of a new candidate. It iscontemplated that many of the approaches useful for designing moleculesde novo may also be useful for modifying existing molecules.

[0356] It is contemplated that knowledge of the spatial relationshipbetween a protein biosynthesis inhibitor, for example, an antibiotic,and its respective binding site within a ribosome permits the design ofmodified inhibitors that may have better binding properties, forexample, higher binding affinity and/or specificity, relative to themolecule from which it was derived. Alternatively, knowledge ofinhibitor contact sites within a ribosome permits the synthesis of a newmolecule that contain, for example, a portion of a first molecule thatbinds to the contact site and another portion that contributesadditional functionality.

[0357] It is contemplated that a variety of modified molecules (forexample, modified antibiotics) may be designed using the atomicco-ordinates provided herein. For example, it is contemplated that byknowing the spatial relationship of one or more of antibiotics relativeto the large ribosomal subunit it is possible to generate newantibiotic-based molecules. The atomic co-ordinates of each antibioticrelative to the large ribosomal subunit provides information on whatportions of the ribosome or ribosomal subunit and the antibiotic contactone another. Accordingly, from this information the skilled artisan maynot only identify contact locations within the ribosome that can be usedfor de novo drug design, as discussed above, but also may identifyportions of an antibiotic that can act as a ribosome binding domain.

[0358] Based on the information provided herein, the skilled artisan mayreadily identify and produce hybrid antibiotics that comprise a ribosomebinding domain of a first antibiotic and a ribosome binding domain of asecond, different antibiotic. The resulting hybrid antibioticspreferably bind to each of respective contact locations within theribosomal subunit simultaneously. The atomic co-ordinates providedherein permit the skilled artisan to identify candidate antibiotics thatmay be used as templates in the synthesis of a hybrid, and also providesteric information necessary to produce linking chemistries such thateach ribosome binding domain is properly orientated relative to itsrespective contact site. As a result, it is contemplated that theskilled artisan may produce a hybrid antibiotic that binds to a ribosomeor ribosomal subunit with a higher affinity and/or have higher proteinsynthesis inhibitory activity than either of the individual templateantibiotics used to generate the hybrid. Alternatively, the hybridantibiotic may overcome resistance phenotypes that may have developedagainst either of the template antibiotics. For example, the proximityof the site occupied by the disaccharide moiety of carbomycin to thesite filled by anisomycin suggests that a hybrid compound includingportions of both carbomycin and anisomycin may be an effective inhibitorof protein synthesis.

[0359] Furthermore, the atomic co-ordinates provided herein permit theskilled artisan to use the information pertaining to identify a ribosomebinding domain and to design other types of protein synthesisinhibitors. For example, with an understanding of the ribosome contactregion and the surrounding environment, the skilled artisan can providenovel molecules, a portion of which is based upon the antibiotic bindingregion (binding domain) and another portion of which (effector domain)can be designed as a novel space filling domain that sterically inhibitsor disrupts protein biosynthesis within the ribosome or secretionthrough the polypeptide exit tunnel. For example, the skilled artisanmay combine the ribosome binding region of the antibiotic, tylosin,which binds to one side of the polypeptide exit tunnel close to thepeptidyl transferase site, with a novel chemical moiety bulky enough toblock the polypeptide exit tunnel. However, it is contemplated that theskilled artisan may take advantage of one or more of the many of theantibiotic contact regions disclosed herein to design entirely newbinding and effector domains.

[0360] Furthermore, the present invention permits the skilled artisan todesign molecules, for example, selective protein synthesis inhibitorsthat are tailored to be more potent with respect to ribosomes of atarget organism, for example, a pathogen such a microbe, and lesspotent, i.e., less toxic, to ribosomes of a non target organism, forexample, host organism such as a human. Also, the invention permits theskilled artisan to use the atomic co-ordinates and structures of thelarge ribosomal subunit and its complexes with protein synthesisinhibitors to design modifications to starting compounds, such as anantibiotic, that will bind more tightly to a target ribosome (e.g., the50S ribosomal subunit of bacteria) and less tightly to a non-targetedribosome (e.g., human 60S ribosomal subunit).

[0361] The structure of a complex between the large ribosomal subunitand the starting compound (e.g., tylosin or another protein synthesisinhibitor) can also be used to guide the modification of that compoundto produce new compounds that have other desirable properties for theapplicable industrial and other uses (e.g., as pharmaceuticals,herbicides or insecticides), such as chemical stability, solubility ormembrane permeability.

[0362] A variety of antibiotics bind the large ribosomal subunit anddisrupt protein synthesis and include members of antibiotic familieswhich include, for example, chloramphenicols, macrolides, lincosamides,streptogramins, althiomycins, oxazolinones, nucleotide analogs,thiostreptons, peptides, glutarimides, and trichothecenes.

[0363] Members of the chloramphenicol family include, for example,Chloramphenicol and Iodoamphenicol. Members of the macrolide familyinclude, for example, Biaxin (Clarithromycin), Zithromax(Azithromycins), Ketek (Telithromycin; ketolide), ABT-773, Tylosin,Spiramycin I, Spiramycin II, Spiramycin III, Erythromycin A, CarbomycinA, Telithromycin, Methymycin, Narbomycin, Lankamycin, Oleandomycin,Megalomycin, Chalcomycin, Niddamycin, Leucomycin, Angolamycin, andRelomycin. Members of the licosamide family include, for example,Clindamycin and Lincomycin. Members of the streptogramin family include,for example, Streptogramin A, Streptogramin B, Ostreogrycin G, Synercid,Virginiamycin S1, Virginiamycin S2, Virginiamycin S3, Virginiamycin S4,Vemamycin B, Vernamycin C, Patricin A, and Patricin B. A member of thealthiomycin family, includes, for example, Althiomycin. A member of theoxazolidine family, includes, for example, Linezolid. Members of thefamily of nucleotide analogs include, for example, Sparsomycin,Puromycin, Anisomycin, and Blasticidin S. Members of the thiostreptonfamily include, for example, Thiostrepton, Siomycin, Sporangiomycin, andThiopeptin. Members of the peptide family include, for example,Viomycin, Capreomycin IA, Capreomycin IB, Capreomycin IIA, andCapreomycin IIB. Members of the glutarimide family include, for example,Cycloheximide, Streptovitacins, Streptimidone, Inactone, Actiphenol.Members of the trichothecene family include, for example, Trichodermin,Trichodermol, Trichodermone, Vomitoxin, T-2 toxin, Trichothecin,Nivalenol, and Verrucarin A.

[0364] Inhibitors can be diffused into or soaked with the stabilizedcrystals of the large ribosomal subunit as described in Example 3 toform a complex with the large ribosomal subunit for collecting X-raydiffraction data. Alternatively, the inhibitors can be co-crystallizedwith the large ribosomal subunit by mixing the inhibitor with the largeribosomal subunit before precipitation with high salt.

[0365] Starting with the structure of the ribosome from H. marismortui,the structure of the ribosome from a non-targeted organism (for example,the human 60S ribosomal subunit) can be constructed by homologymodeling, i.e., by changing the structure of residues at a target siteof interest for the residues at the same positions in of the non-targetribosome. This is done computationally by removing the side chains fromthe ribosome of known structure and replacing them by the side chains ofthe unknown structure put in sterically plausible positions. In thisway, it can be understood how the shapes of the target sites within thetargeted and non-targeted ribosomes differ. This process, therefore,provides information concerning how a molecule that binds the targetsite can be chemically altered in order to produce molecules that willbind tightly and specifically to the targeted ribosome but willsimultaneously be prevented from binding to the non-targeted ribosome.Likewise, knowledge of portions of the bound molecules that face thesolvent permit introduction of other functional groups for additionalpharmaceutical purposes. The process of homology structure modeling canalso be used to understand the mechanisms whereby mutant ribosomesbecome resistant to the effects of pharmaceuticals or pesticides, suchas herbicides or insecticides. Furthermore, with knowledge of theportions of the ribosomal subunit that participates in drug resistance,the skilled artisan may design new molecules that overcome the problemof drug resistance.

[0366] The use of homology structure modeling to design molecules thatbind more tightly to the target ribosome than to the non-target ribosomehas wide-spread applicability. The methods outlined herein can be usedto control any targeted organism, for example, a pathogen, by designingmolecules that inhibit large ribosomal subunits of the targetedorganisms while failing to inhibit the 50S or 60S ribosomal subunit ofthe non-targeted organism, for example, a host, to the same extent ornot at all. The molecules identified or prepared by the methods of thepresent invention can be used to control the targeted organisms whilecausing the non-targeted organism little or no adverse effects. Thus,the molecules identified or developed using the methods of the presentinvention can be designed so that their administration kills the targetorganisms or inhibits some aspect of the biological functions of thetarget organisms while failing to have a similar effect on thenon-targeted organism. The adverse effects of the agent on the targetedorganisms may include, but are not limited to, death of the targetorganism; slowing growth rates; slowing or eliminating passage from onegrowth phase to another (e.g., extending the larval growth stage);slowing or eliminating reproduction, decreasing or preventing mating,decreasing or eliminating offspring production, limiting or eliminatingtarget organism weight gains; decreasing or eliminating feeding abilityand behaviors; and disrupting cellular, tissue and/or organ functions.

[0367] The novel agents contemplated by the present invention can beuseful as herbicides, pesticides (e.g., insecticides, nematocides,rodenticides, etc.), miticides, or antimicrobial agents (e.g.,antifungals, antibacterials, antiprotozoals, etc.) to target specificorganisms. For example, the novel agents can target animal and plantparasitic nematodes, prokaryotic organisms (disease causing microbes),and eukaryotic multicellular pests. Specific examples of multicellularpests include, but are not limited to, insects, fungi, bacteria,nematodes, mites and ticks, protozoan pathogens, animal-parasitic liverflukes, and the like.

[0368] Herbicides, pesticides, miticides, and antimicrobial agents thatinhibit protein synthesis by interacting with ribosomes are known to theskilled artisan. A few examples are discussed below. These known agentscan be modified to obtain novel agents by using computer modelingtechniques and knowledge of the structure of ribosomes and ribosomalsubunits and the structure of ribosome/agent and ribosomal subunit/agentcomplexes.

[0369] The ketolide ABT-773 binds ribosomes tighter than erythromycin inS. pneumoniae and is able to defeat macrolide resistance in bacteria(Capobianco et al. (2000) Antimicrob Agents Chemother 44(6), 1562-1567).The tools and methodologies of the present invention can be used toobtain erythromycin derivatives that bind the ribosomes or ribosomalsubunits of target bacteria more tightly than they bind the ribosomesand ribosomal subunits of non-target animals. The target bacteria can beany infectious bacteria, particularly S. pneumoniae, and even moreparticularly erythromycin-resistant S. pneumoniae. The non-targetanimals can be any animal, particularly mammals, and even moreparticularly humans.

[0370] Examples of antibiotics that are inhibitors of protein synthesisinclude, but are not limited to, puromycin, cycloheximide,chloramphenicol, tetracycline, and streptomycin (Heldt, (1996) PlantBiochemistry and Molecular Biology 21.2: 458-464). Puromycin, asdiscussed earlier, binds as an analogue of an aminoacyl-tRNA to theA-site and is added to nascent peptide chains, its weak associate withthe ribosome prevents further elongation steps in prokaryotes andeukaryotes. Cycloheximide inhibits peptidyl transferase in eukaryoticribosomes. Chloramphenicol inhibits peptidyl transferase in prokaryoticribosomes. Tetracycline binds to the 30S subunit and inhibits thebinding of aminoacyl-tRNA to prokaryotic ribosomes much more than toeukaryotic ones. Streptomycin interacts with 30S ribosomes which resultsin an incorrect recognition of mRNA sequences and thus inhibitsinitiation in prokaryotic ribosomes. U.S. Pat. No. 5,801,153 disclosesantibiotics against pathogens. Aminoglycosides are examples ofantibacterial antibiotics that appear to inhibit protein synthesis.However, there is a limitation to their use because of their ototoxicand nephrotoxic properties. Amikacin sulfate, Framycetin sulfate,Gentamycin sulfate, Kanamycin sulfate, Neomycin sulfate, Netilmicinsulfate, Paromomycin sulfate, Sissomycin sulfate, Tobramycin, Vancomycinhydrochloride, and Viomycin sulfate are the members of theaminoglycoside family. The tools and methodologies of the presentinvention can be used to obtain derivatives of any antibiotic of choiceso that they inhibit the protein synthesis of target organisms to agreater degree than they inhibit the protein synthesis of non-targetorganisms, such as humans.

[0371] Examples of targeted and non-targeted organisms include, but arenot limited to, those provided in Table 18. TABLE 18 Examples of Classesof Molecules which can be Identified and/or Developed by the Methods ofthe Invention and Applicable Target/Non-Target Organisms. Type ofMolecule Target Organisms Non-Target Organisms Herbicides Dicotyledonousplants Monocotyledonous plants Herbicides Grasses Soybeans, potatoes,coffee Insecticides Flies, Mites Honey bees Pesticides Ticks DeerPesticides Lice Birds Miticides Parasitic mites (mange) DogsAntimicrobial Streptococcus pneumoniae Humans Agents (Antibacterials)Antimicrobial Clostridium difficile Escherichia coli Agents(Antibacterials) Antimicrobial Erysiphe graminis Barley Agents(Antifungals) Antimicrobial Toxoplasma gondii Animals Agents(Antiprotozoals) Poisons Rats Dogs, cats, humans (Rodentcides)

[0372] It is contemplated that the tools and methodologies of thepresent invention can be used to obtain inhibitors of protein synthesisof target insects, such as bollworms and mosquitoes, more than theyinhibit the protein synthesis of non-target insects, such as beetles ofthe family Coccinellidae (e.g., ladybugs) and Apis mellifera (honeybees). Other possible target insects include, but are not limited to,insects selected from the orders Coleoptera (beetles), Diptera (flies,mosquitoes), Hymenoptera (wasps, ants, sawflies), Lepidoptera(butterflies and moths), Mallophaga (lice), Homoptera (whiteflies,aphids), Hemiptera (bugs), Orthroptera (locusts, cockroaches),Thysanoptera (thrips), Dermaptera (earwigs), Isoptera, Anoplura,Siphonaptera, and Trichoptera (caddis flies).

[0373] Furthermore, it is contemplated that the tools and methodologiesof the present invention can be used to obtain inhibitors of proteinsynthesis of target plants which inhibit protein synthesis of the targetplants more than they inhibit the protein synthesis of non-target plantsand animals. The target plants can be any unwanted plant species,particular weeds, and even more particularly noxious weeds. Whether ornot a particular plant is considered a weed will depend upon the contextin which it is growing. For example, unwanted Zea mays (corn) plantsgrowing in a Glycine max (soybean) field could be considered unwantedweeds. Examples of weeds which are likely target plants include, but arenot limited to, Allium vineale (wild garlic), Bromus tectorum (downybrome), Triticum cylindricum (jointed goatgrass), Amaranthus spp.(pigsweed), Chenopodium album (lambsquarters), Avena fatua (wild oats),B. secalinus (cheat), Echinochloa crus-galli (barnyardgrass), Alopecurusmyosuroides (blackgrass), Setaria faberii (giant foxtail), Xanthiumstrumarium (common cocklebur), Ambrosia artemisiifolia (common ragweed),and Ipomoea spp. (morning glories). The non-target organisms can be anyplant, particularly any desirable plant, and even more particularly anycrop plant. The non-target organisms can also be any animals,particularly mammals, and even more particularly humans. In onepreferred embodiment, the tools and methodologies of the presentinvention can be used to produce protein synthesis inhibitors which killor injure one or more noxious weed species but fail to harm non-targetplants and animals.

[0374] Target bacteria of interest include, but are not limited to,Staphylococcus aureus, Streptococcus pyogenes, Streptococcus agalactiae,Streptococcus bovis, Streptococcus pneumoniae, Moraxella catarrhalis,Neisseria gonorrhoeae, Neisseria meningitides, Bacillus anthracis,Corynebacterium diphtheriae, Listeria monocytogenes, Erysipelothrixrhusiopathiae, Clostridium perfringens, Clostridium tetani, Clostridiumdifficile, Eschericia coli, Proteus mirabilis, Psuedomonas aeruginosa,Klebsiella pneumoniae, Haemophilus influenzae, Haemophilus ducreyi,Yersinia pestis, Yersinia enterocolitica, Francisella tularensis,Pasteurella multocida, Vibrio cholerae, Flavobacterium meningosepticum,Pseudomonas mallei, Pseudomonas pseudomallei, Campylobacter jejuni,Campylobacter fetus, Fusobacterium nucleatum, Calymmatobacteriumgranulomatis, Streptobacillus moniliformis, Legionella pneumophila,Mycobacterium avium-intracellulare, Mycobacterium tuberculosis,Mycobacterium leprae, Treponema pallidum, Treponema pertenue, Borreliaburgdorferi, Borrelia recurrentis, Actinomyces isrealii, Nocardiaasteroides, Ureaplasma urealyticum, Mycoplasma pneumoniae, Chlamydiapsittaci, Chlamydia trachomatis, Chlamydia pnemoniae, Pneumocystiscarinii, Coccidioides immitis, Histoplasma capsulatum, Blastomycesdermatitidis, Paracoccidioides brasiliensis, Sporothrix schenckii,Cryptococcus neoformans.

[0375] Once a candidate molecule has been designed or selected by theabove methods, the affinity with which that molecule may bind to theribosome or ribosomal subunit may be tested and optimized bycomputational evaluation and/or by testing biological activity aftersynthesizing the compound. Candidate molecules may interact with theribosomes or ribosomal subunits in more than one conformation each ofwhich has a similar overall binding energy. In those cases, thedeformation energy of binding may be considered to be the differencebetween the energy of the free molecule and the average energy of theconformations observed when the molecule binds to the ribosomes orribosomal subunits, more preferably to the large ribosomal subunits, andeven more preferably to the 50S ribosomal subunits.

[0376] A molecule designed or selected as binding to a ribosome orribosomal subunit may be further computationally optimized so that inits bound state it preferably lacks repulsive electrostatic interactionwith the target region. Such non-complementary (e.g., electrostatic)interactions include repulsive charge-charge, dipole-dipole andcharge-dipole interactions. Specifically, the sum of all electrostaticinteractions between the inhibitor and the enzyme when the inhibitor isbound to the ribosome or the ribosomal subunit, preferably make aneutral or favorable contribution to the enthalpy of binding. Weakbinding compounds can also be designed by these methods so as todetermine SAR.

[0377] Specific computer programs that can evaluate a compounddeformation energy and electrostatic interaction are available in theart. Examples of suitable programs include: Gaussian 92, revision C (M.J. Frisch, Gaussian, Inc., Pittsburgh, PA.); AMBER, version 4.0 (P. A.Kollman, University of California at San Francisco, Calif.);QUANTA/CHARMM (Molecular Simulations, Inc., Burlington, Mass.); andInsight II/Discover (Biosysm Technologies Inc., San Diego, Calif.).These programs may be implemented, for instance, using a SiliconGraphics workstation, IRIS 4D/35 or IBM RISC/6000 workstation model 550.Other hardware systems and software packages are known to those skilledin the art.

[0378] Once a molecule of interest has been selected or designed, asdescribed above, substitutions may then be made in some of its atoms orside groups in order to improve or modify its binding properties.Generally, initial substitutions are conservative, i.e., the replacementgroup will approximate the same size, shape, hydrophobicity and chargeas the original group. It should, of course, be understood thatcomponents known in the art to alter conformation should be avoided.Such substituted chemical compounds may then be analyzed for efficiencyof fit to the ribosome or ribosomal subunit by the same computer methodsdescribed in detail, above.

[0379] In addition, the actual ribosome-related ligands, complexes ormimetics may be crystallized and analyzed using X-ray diffraction. Thediffraction pattern co-ordinates are similarly used to calculate thethree-dimensional interaction of a ligand and the ribosome, ribosomalsubunit, or a mimetic, in order to confirm that the ligand binds to, orchanges the conformation of, a particular site on the ribosome orribosomal subunit, or where the mimetic has a similar three-dimensionalstructure to that of a ribosome, ribosomal subunit or a fragmentthereof.

[0380] 3. Synthesis of Lead Molecules

[0381] A lead molecule of the present invention can be, but is notlimited to, at least one selected from a lipid, nucleic acid, peptide,small organic or inorganic molecule, chemical compound, element,saccharide, isotope, carbohydrate, imaging agent, lipoprotein,glycoprotein, enzyme, analytical probe, and an antibody or fragmentthereof, any combination of any of the foregoing, and any chemicalmodification or variant of any of the foregoing. In addition, a leadmolecule may optionally comprise a detectable label. Such labelsinclude, but are not limited to, enzymatic labels, radioisotope orradioactive compounds or elements, fluorescent compounds or metals,chemiluminescent compounds and bioluminescent compounds. Well knownmethods may be used for attaching such a detectable label to a leadmolecule.

[0382] Methods useful for synthesizing lead molecules such as lipids,nucleic acids, peptides, small organic or inorganic molecules, chemicalcompounds, elements, saccharides, isotopes, carbohydrates, imagingagents, lipoproteins, glycoproteins, enzymes, analytical probes,antibodies, and antibody fragments are well known in the art. Suchmethods include the traditional approach of synthesizing one such leadmolecule, such as a single defined peptide, at a time, as well ascombined synthesis of multiple lead molecules in a one or morecontainers. Such multiple lead molecules may include one or morevariants of a previously identified lead molecule. Methods for combinedsynthesis of multiple lead molecules are particularly useful inpreparing combinatorial libraries, which may be used in screeningtechniques known in the art.

[0383] By way of example, it is well known in the art that multiplepeptides and oligonucleotides may be simultaneously synthesized. Leadmolecules that are small peptides up to 50 amino acids in length, may besynthesized using standard solid-phase peptide synthesis procedures, forexample, procedures similar to those described in Merrifield (1963) J.Am. Chem. Soc., 85: 2149. For example, during synthesis, N-α-protectedamino acids having protected side chains are added stepwise to a growingpolypeptide chain linked by its C-terminal end to an insoluble polymericsupport, e.g., polystyrene beads. The peptides are synthesized bylinking an amino group of an N-α-deprotected amino acid to an α-carboxygroup of an N-α-protected amino acid that has been activated by reactingit with a reagent such as dicyclohexylcarbodiimide. The attachment of afree amino group to the activated carboxyl leads to peptide bondformation. The most commonly used N-α-protecting groups include Bocwhich is acid labile and Fmoc which is base labile.

[0384] Briefly, the C-terminal N-α-protected amino acid is firstattached to the polystyrene beads. Then, the N-α-protecting group isremoved. The deprotected α-amino group is coupled to the activatedα-carboxylate group of the next N-α-protected amino acid. The process isrepeated until the desired peptide is synthesized. The resultingpeptides are cleaved from the insoluble polymer support and the aminoacid side chains deprotected. Longer peptides, for example greater thanabout 50 amino acids in length, typically are derived by condensation ofprotected peptide fragments. Details of appropriate chemistries, resins,protecting groups, protected amino acids and reagents are well known inthe art and so are not discussed in detail herein. See for example,Atherton et al. (1963) Solid Phase Peptide Synthesis: A PracticalApproach (IRL Press), and Bodanszky (1993) Peptide Chemistry, APractical Textbook, 2nd Ed. Springer-Verlag, and Fields et al. (1990)Int. J. Peptide Protein Res. 35:161-214.

[0385] Purification of the resulting peptide is accomplished usingconventional procedures, such as preparative HPLC, e.g., gel permeation,partition and/or ion exchange chromatography. The choice of appropriatematrices and buffers are well known in the art and so are not describedin detail herein.

[0386] It is contemplated that a synthetic peptide in accordance withthe invention may comprise naturally occurring amino acids, unnaturalamino acids, and/or amino acids having specific characteristics, suchas, for example, amino acids that are positively charged, negativelycharged, hydrophobic, hydrophilic, or aromatic. As used herein, the term“naturally occurring amino acids” refers to the L-isomers of amino acidsnormally found in proteins. The predominant naturally occurring aminoacids are glycine, alanine, valine, leucine, isoleucine, serine,methionine, threonine, phenylalanine, tyrosine, tryptophan, cysteine,proline, histidine, aspartic acid, asparagine, glutamic acid, glutamine,arginine, and lysine. Unless specifically indicated, all amino acids arereferred to in this application are in the L-form. Furthermore, as usedherein, the term “unnatural amino acids” refers to amino acids that arenot naturally found in proteins. For example, selenomethionine.

[0387] Amino acids that are “positively charged” include any amino acidhaving a positively charged side chain under normal physiologicalconditions. Examples of positively charged naturally occurring aminoacids include, for example, arginine, lysine, and histidine. Conversely,amino acids that are “negatively charged” include any amino acid havinga negatively charged side chains under normal physiological conditions.Examples of negatively charged naturally occurring amino acids include,for example, aspartic acid and glutamic acid.

[0388] As used herein, the term “hydrophobic amino acid” includes anyamino acids having an uncharged, nonpolar side chain that is relativelyinsoluble in water. Examples of naturally occurring hydrophobic aminoacids include, for example, alanine, leucine, isoleucine, valine,proline, phenylalanine, tryptophan, and methionine. In addition, as usedherein, the term “hydrophilic amino acid” refers to any amino acidshaving an uncharged, polar side chain that is relatively soluble inwater. Examples of naturally occurring hydrophilic amino acids include,for example, serine, threonine, tyrosine, asparagine, glutamine andcysteine.

[0389] Finally, as used herein, the term “aromatic” refers to amino acidresidues which side chains have delocalized conjugated system. Examplesof aromatic residues include, for example, phenylalanine, tryptophan,and tyrosine.

[0390] With regard to the production of non-peptide small organicmolecules which act as a ligand in the present invention, thesemolecules can be synthesized using standard organic chemistries wellknown and thoroughly documented in the patent and other literatures.

[0391] Many of the known methods useful in synthesizing lead of thepresent invention may be automated, or may otherwise be practiced on acommercial scale. As such, once a lead molecule has been identified ashaving commercial potential, mass quantities of that molecule may easilybe produced.

[0392] 4. Characterization of Molecules

[0393] Molecules designed, selected and/or optimized by methodsdescribed above, once produced, may be characterized using a variety ofassays known to those skilled in the art to determine whether thecompounds have biological activity. For example, the molecules may becharacterized by conventional assays, including but not limited to thoseassays described below, to determine whether they have a predictedactivity, binding activity and/or binding specificity.

[0394] Furthermore, high-throughput screening may be used to speed upanalysis using such assays. As a result, it may be possible to rapidlyscreen new molecules for their ability to interact with a ribosome orribosomal subunit using the tools and methods of the present invention.General methodologies for performing high-throughput screening aredescribed, for example, in Devlin, (1998), High Throughput Screening,Marcel Dekker; and U.S. Pat. No. 5,763,263. High-throughput assays canuse one or more different assay techniques including, but not limitedto, those described below.

[0395] (1) Surface Binding Studies. A variety of binding assays may beuseful in screening new molecules for their binding activity. Oneapproach includes surface plasmon resonance (SPR) which can be used toevaluate the binding properties molecules of interest with respect to aribosome, ribosomal subunit or a fragment thereof.

[0396] SPR methodologies measure the interaction between two or moremacromolecules in real-time through the generation of aquantum-mechanical surface plasmon. One device, (BlAcore Biosensor RTMfrom Pharmacia Biosensor, Piscatawy, N.J.) provides a focused beam ofpolychromatic light to the interface between a gold film (provided as adisposable biosensor “chip”) and a buffer compartment that can beregulated by the user. A 100 nm thick “hydrogen” composed ofcarboxylated dextran which provides a matrix for the covalentimmobilization of analytes of interest is attached to the gold film.When the focused light interacts with the free electron cloud of thegold film, plasmon resonance is enhanced. The resulting reflected lightis spectrally depleted in wavelengths that optimally evolved theresonance. By separating the reflected polychromatic light into itscomponent wavelengths (by means of a prism), and determining thefrequencies which are depleted, the BlAcore establishes an opticalinterface which accurately reports the behavior of the generated surfaceplasmon resonance. When designed as above, the plasmon resonance (andthus the depletion spectrum) is sensitive to mass in the evanescentfield (which corresponds roughly to the thickness of the hydrogel). Ifone component of an interacting pair is immobilized to the hydrogel, andthe interacting partner is provided through the buffer compartment, theinteraction between the two components can be measured in real timebased on the accumulation of mass in the evanescent field and itscorresponding effects of the plasmon resonance as measured by thedepletion spectrum. This system permits rapid and sensitive real-timemeasurement of the molecular interactions without the need to labeleither component.

[0397] (2) Immunodiagnostics and Immunoassays. These are a group oftechniques that can be used for the measurement of specific biochemicalsubstances, commonly at low concentrations in complex mixtures such asbiological fluids, that depend upon the specificity and high affinityshown by suitably prepared and selected antibodies for theircomplementary antigens. A substance to be measured must, of necessity,be antigenic - either an immunogenic macromolecule or a haptenic smallmolecule. To each sample a known, limited amount of specific antibody isadded and the fraction of the antigen combining with it, often expressedas the bound:free ratio, is estimated, using as indicator a form of theantigen labeled with radioisotope (radioimmunoassay), fluorescentmolecule (fluoroimmunoassay), stable free radical (spin immunoassay),enzyme (enzyme immunoassay), or other readily distinguishable label.

[0398] Antibodies can be labeled in various ways, including:enzyme-linked immunosorbent assay (ELISA); radioimmuno assay (RIA);fluorescent immunoassay (FIA); chemiluminescent immunoassay (CLIA); andlabeling the antibody with colloidal gold particles (immunogold).

[0399] Common assay formats include the sandwich assay, competitive orcompetition assay, latex agglutination assay, homogeneous assay,microtitre plate format and the microparticle-based assay.

[0400] (3) Enzyme-linked immunosorbent assay (ELISA). ELISA is animmunochemical technique that avoids the hazards of radiochemicals andthe expense of fluorescence detection systems. Instead, the assay usesenzymes as indicators. ELISA is a form of quantitative immunoassay basedon the use of antibodies (or antigens) that are linked to an insolublecarrier surface, which is then used to “capture” the relevant antigen(or antibody) in the test solution. The antigen-antibody complex is thendetected by measuring the activity of an appropriate enzyme that hadpreviously been covalently attached to the antigen (or antibody).

[0401] General methods and compositions for practicing ELISA aredescribed, for example, in Crowther (1995) ELISA—Theory and Practice(Methods in Molecular Biology), Humana Press; Challacombe and Kemeny,(1998) ELISA and Other Solid Phase Immunoassays—Theoretical andPractical Aspects, John Wiley; Kemeny, (1991) A Practical Guide toELISA, Pergamon Press; Ishikawa, (1991) Ultrasensitive and Rapid EnzymeImmunoassay (Laboratory Techniques in Biochemistry and MolecularBiology) Elsevier.

[0402] (4) Colorimetric Assays. Colorimetry is any method ofquantitative chemical analysis in which the concentration or amount of acompound is determined by comparing the color produced by the reactionof a reagent with both standard and test amounts of the compound, oftenusing a calorimeter. A colorimeter is a device for measuring colorintensity or differences in color intensity, either visually orphotoelectrically.

[0403] Standard colorimetric assays of beta-galactosidase enzymaticactivity are well known to those skilled in the art (see, for example,Norton et al. (1985) Mol. Cell. Biol. 5: 281-290). A colorimetric assaycan be performed on whole cell lysates usingO-nitrophenyl-β-D-galactopyranoside (ONPG, Sigma) as the substrate in astandard calorimetric beta-galactosidase assay (Sambrook et al. (1989)Molecular Cloning—A Laboratory Manual, Cold Spring Harbor LaboratoryPress). Automated calorimetric assays are also available for thedetection of β-galactosidase activity, as described in U.S. Pat. No.5,733,720.

[0404] (5) Immunofluorescence Assays. Immunofluorescence orimmunofluorescence microscopy is a technique in which an antigen orantibody is made fluorescent by conjugation to a fluorescent dye andthen allowed to react with the complementary antibody or antigen in atissue section or smear. The location of the antigen or antibody canthen be determined by observing the fluorescence by microscopy underultraviolet light.

[0405] A general description of immunofluorescent techniques appears forexample, in Knapp et al. (1978) Immunofluorescence and Related StainingTechniques, Elsevier; Allan, (1999) Protein Localization by FluorescentMicroscopy—A Practical Approach (The Practical Approach Series) OxfordUniversity Press; Caul, (1993) Immunofluorescence Antigen DetectionTechniques in Diagnostic Microbiology, Cambridge University Press. Fordetailed explanations of immunofluorescent techniques applicable to thepresent invention, see, for example, U.S. Pat. No. 5,912,176; U.S. Pat.No. 5,869,264; U.S. Pat. No. 5,866,319; and U.S. Pat. No. 5,861,259.

[0406] (6) Fluorescence Polarization. Fluorescence polarization (FP) isa measurement technique that can readily be applied to protein-proteinand protein-ligand interactions in order to derive IC₅₀s and Kds of theassociation reaction between two molecules. In this technique one of themolecules of interest is conjugated with a fluorophore. This isgenerally the smaller molecule in the system (in this case, the moleculeof interest). The sample mixture, containing both the ligand-probeconjugate and the ribosome, ribosomal subunit or fragment thereof, isexcited with vertically polarized light. Light is absorbed by the probefluorophores, and re-emitted a short time later. The degree ofpolarization of the emitted light is measured. Polarization of theemitted light is dependent on several factors, but most importantly onviscosity of the solution and on the apparent molecular weight of thefluorophore. With proper controls, changes in the degree of polarizationof the emitted light depends only on changes in the apparent molecularweight of the fluorophore, which in-turn depends on whether theprobe-ligand conjugate is free in solution, or is bound to a receptor.Binding assays based on FP have a number of important advantages,including the measurement of IC₅₀s and Kds under true homogenousequilibrium conditions, speed of analysis and amenity to automation, andability to screen in cloudy suspensions and colored solutions.

[0407] (7) Protein Synthesis. It is contemplated that, in addition tocharacterization by the foregoing biochemical assays, the molecule ofinterest may also be characterized as a modulator (for example, aninducer of protein synthesis or an inhibitor of protein synthesis) ofthe functional activity of the ribosome or ribosomal subunit.

[0408] Inhibitors of protein synthesis may be assayed on the cellularlevel. For example, molecules of interest can be assayed for inhibitoryaction against organisms, for example, micro-organism, by growing themicro-organism of interest in media either containing or lacking themolecule of interest. Growth inhibition may be indicative that themolecule may be acting as a protein synthesis inhibitor.

[0409] Furthermore, more specific protein synthesis inhibition assaysmay be performed by administering the compound to a whole organism,tissue, organ, organelle, cell, a cellular or subcellular extract, or apurified ribosome preparation and observing its pharmacological andinhibitory properties by determining, for example, its inhibitionconstant (IC₅₀) for inhibiting protein synthesis. Incorporation of ³Hleucine or ³⁵S methionine, or similar experiments can be performed toinvestigate protein synthesis activity.

[0410] A change in the amount or the rate of protein synthesis in thecell in the presence of a molecule of interest indicates that themolecule is an inducer of protein synthesis. A decrease in the rate orthe amount of protein synthesis indicates that the molecule is ainhibitor of protein synthesis.

[0411] H. Drug Formulation and Administration

[0412] It is contemplated that once identified, the active molecules ofthe invention may be incorporated into any suitable carrier prior touse. More specifically, the dose of active molecule, mode ofadministration and use of suitable carrier will depend upon the targetand non-target organism of interest.

[0413] It is contemplated that with regard to mammalian recipients, thecompounds of interest may be administered by any conventional approachknown and/or used in the art. Thus, as appropriate, administration canbe oral or parenteral, including intravenous and intraperitoneal routesof administration. In addition, administration can be by periodicinjections of a bolus, or can be made more continuous by intravenous orintraperitoneal administration from a reservoir which is external (e.g.an intrvenous bag). In certain embodiments, the compounds of theinvention can be therapeutic-grade. That is, certain embodiments complywith standards of purity and quality control required for administrationto humans. Veterinary applications are also within the intended meaningas used herein.

[0414] The formulations, both for veterinary and for human medical use,of the drugs according to the present invention typically include suchdrugs in association with a pharmaceutically acceptable carriertherefore and optionally other therapeutic ingredient(s). The carrier(s)should be “acceptable” in the sense of being compatible with the otheringredients of the formulations and not deleterious to the recipientthereof. Pharmaceutically acceptable carriers, in this regard, areintended to include any and all solvents, dispersion media, coatings,antibacterial and antifungal agents, isotonic and absorption delayingagents, and the like, compatible with pharmaceutical administration. Theuse of such media and agents for pharmaceutically active substances isknown in the art. Except insofar as any conventional media or agent isincompatible with the active compound, use thereof in the compositionsis contemplated. Supplementary active compounds (identified or designedaccording to the invention and/or known in the art) also can beincorporated into the compositions. The formulations may conveniently bepresented in dosage unit form and may be prepared by any of the methodswell known in the art of pharmacy/microbiology. In general, someformulations are prepared by bringing the drug into association with aliquid carrier or a finely divided solid carrier or both, and then, ifnecessary, shaping the product into the desired formulation.

[0415] A pharmaceutical composition of the invention should beformulated to be compatible with its intended route of administration.Examples of routes of administration include oral or parenteral, e.g.,intravenous, intradermal, inhalation, transdermal (topical),transmucosal, and rectal administration. Solutions or suspensions usedfor parenteral, intradermal, or subcutaneous application can include thefollowing components: a sterile diluent such as water for injection,saline solution, fixed oils, polyethylene glycols, glycerine, propyleneglycol or other synthetic solvents; antibacterial agents such as benzylalcohol or methyl parabens; antioxidants such as ascorbic acid or sodiumbisulfite; chelating agents such as ethylenediaminetetraacetic acid;buffers such as acetates, citrates or phosphates and agents for theadjustment of tonicity such as sodium chloride or dextrose. pH can beadjusted with acids or bases, such as hydrochloric acid or sodiumhydroxide.

[0416] Useful solutions for oral or parenteral administration can beprepared by any of the methods well known in the pharmaceutical art,described, for example, in Remington's Pharmaceutical Sciences,(Gennaro, A., ed.), Mack Pub., (1990). Formulations for parenteraladministration can also include glycocholate for buccal administration,methoxysalicylate for rectal administration, or cutric acid for vaginaladministration. The parenteral preparation can be enclosed in ampoules,disposable syringes or multiple dose vials made of glass or plastic.Suppositories for rectal administration also can be prepared by mixingthe drug with a non-irritating excipient such as cocoa butter, otherglycerides, or other compositions which are solid at room temperatureand liquid at body temperatures. Formulations also can include, forexample, polyalkylene glycols such as polyethylene glycol, oils ofvegetable origin, hydrogenated naphthalenes, and the like. Formulationsfor direct administration can include glycerol and other compositions ofhigh viscosity. Other potentially useful parenteral carriers for thesedrugs include ethylene-vinyl acetate copolymer particles, osmotic pumps,implantable infusion systems, and liposomes. Formulations for inhalationadministration can contain as excipients, for example, lactose, or canbe aqueous solutions containing, for example, polyoxyethylene-9-laurylether, glycocholate and deoxycholate, or oily solutions foradministration in the form of nasal drops, or as a gel to be appliedintranasally. Retention enemas also can be used for rectal delivery.

[0417] Formulations of the present invention suitable for oraladministration may be in the form of discrete units such as capsules,gelatin capsules, sachets, tablets, troches, or lozenges, eachcontaining a predetermined amount of the drug; in the form of a powderor granules; in the form of a solution or a suspension in an aqueousliquid or non-aqueous liquid; or in the form of an oil-in-water emulsionor a water-in-oil emulsion. The drug may also be administered in theform of a bolus, electuary or paste. A tablet may be made by compressingor moulding the drug optionally with one or more accessory ingredients.Compressed tablets may be prepared by compressing, in a suitablemachine, the drug in a free-flowing form such as a powder or granules,optionally mixed by a binder, lubricant, inert diluent, surface activeor dispersing agent. Moulded tablets may be made by moulding, in asuitable machine, a mixture of the powdered drug and suitable carriermoistened with an inert liquid diluent.

[0418] Oral compositions generally include an inert diluent or an ediblecarrier. For the purpose of oral therapeutic administration, the activecompound can be incorporated with excipients. Oral compositions preparedusing a fluid carrier for use as a mouthwash include the compound in thefluid carrier and are applied orally and swished and expectorated orswallowed. Pharmaceutically compatible binding agents, and/or adjuvantmaterials can be included as part of the composition. The tablets,pills, capsules, troches and the like can contain any of the followingingredients, or compounds of a similar nature: a binder such asmicrocrystalline cellulose, gum tragacanth or gelatin; an excipient suchas starch or lactose; a disintegrating agent such as alginic acid,Primogel, or corn starch; a lubricant such as magnesium stearate orSterotes; a glidant such as colloidal silicon dioxide; a sweeteningagent such as sucrose or saccharin; or a flavoring agent such aspeppermint, methyl salicylate, or orange flavoring.

[0419] Pharmaceutical compositions suitable for injectable use includesterile aqueous solutions (where water soluble) or dispersions andsterile powders for the extemporaneous preparation of sterile injectablesolutions or dispersion. For intravenous administration, suitablecarriers include physiological saline, bacteriostatic water, CremophorELTM (BASF, Parsippany, N.J.) or phosphate buffered saline (PBS). In allcases, the composition should be sterile and should be fluid to theextent that easy syringability exists. It should be stable under theconditions of manufacture and storage and should be preserved againstthe contaminating action of microorganisms such as bacteria and fungi.The carrier can be a solvent or dispersion medium containing, forexample, water, ethanol, polyol (for example, glycerol, propyleneglycol, and liquid polyetheylene glycol, and the like), and suitablemixtures thereof. The proper fluidity can be maintained, for example, bythe use of a coating such as lecithin, by the maintenance of therequired particle size in the case of dispersion and by the use ofsurfactants. Prevention of the action of microorganisms can be achievedby various antibacterial and antifungal agents, for example, parabens,chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In manycases, it will be preferable to include isotonic agents, for example,sugars, polyalcohols such as manitol, sorbitol, sodium chloride in thecomposition. Prolonged absorption of the injectable compositions can bebrought about by including in the composition an agent which delaysabsorption, for example, aluminum monostearate and gelatin.

[0420] Sterile injectable solutions can be prepared by incorporating theactive compound in the required amount in an appropriate solvent withone or a combination of ingredients enumerated above, as required,followed by filtered sterilization. Generally, dispersions are preparedby incorporating the active compound into a sterile vehicle whichcontains a basic dispersion medium and the required other ingredientsfrom those enumerated above. In the case of sterile powders for thepreparation of sterile injectable solutions, methods of preparationinclude vacuum drying and freeze-drying which yields a powder of theactive ingredient plus any additional desired ingredient from apreviously sterile-filtered solution thereof.

[0421] Formulations suitable for intra-articular administration may bein the form of a sterile aqueous preparation of the drug which may be inmicrocrystalline form, for example, in the form of an aqueousmicrocrystalline suspension. Liposomal formulations or biodegradablepolymer systems may also be used to present the drug for bothintra-articular and ophthalmic administration.

[0422] Formulations suitable for topical administration, including eyetreatment, include liquid or semi-liquid preparations such as liniments,lotions, gels, applicants, oil-in-water or water-in-oil emulsions suchas creams, ointments or pasts; or solutions or suspensions such asdrops. Formulations for topical administration to the skin surface canbe prepared by dispersing the drug with a dermatologically acceptablecarrier such as a lotion, cream, ointment or soap. Particularly usefulare carriers capable of forming a film or layer over the skin tolocalize application and inhibit removal. For topical administration tointernal tissue surfaces, the agent can be dispersed in a liquid tissueadhesive or other substance known to enhance adsorption to a tissuesurface. For example, hydroxypropylcellulose or fibrinogen/thrombinsolutions can be used to advantage. Alternatively, tissue-coatingsolutions, such as pectin-containing formulations can be used.

[0423] For inhalation treatments, inhalation of powder (self-propellingor spray formulations) dispensed with a spray can, a nebulizer, or anatomizer can be used. Such formulations can be in the form of a finepowder for pulmonary administration from a powder inhalation device orself-propelling powder-dispensing formulations. In the case ofself-propelling solution and spray formulations, the effect may beachieved either by choice of a valve having the desired spraycharacteristics (i.e., being capable of producing a spray having thedesired particle size) or by incorporating the active ingredient as asuspended powder in controlled particle size. For administration byinhalation, the compounds also can be delivered in the form of anaerosol spray from pressured container or dispenser which contains asuitable propellant, e.g., a gas such as carbon dioxide, or a nebulizer.

[0424] Systemic administration also can be by transmucosal ortransdermal means. For transmucosal or transdermal administration,penetrants appropriate to the barrier to be permeated are used in theformulation. Such penetrants generally are known in the art, andinclude, for example, for transmucosal administration, detergents, bilesalts, and filsidic acid derivatives. Transmucosal administration can beaccomplished through the use of nasal sprays or suppositories. Fortransdermal administration, the active compounds typically areformulated into ointments, salves, gels, or creams as generally known inthe art.

[0425] The active compounds may be prepared with carriers that willprotect the compound against rapid elimination from the body, such as acontrolled release formulation, including implants and microencapsulateddelivery systems. Biodegradable, biocompatible polymers can be used,such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid,collagen, polyorthoesters, and polylactic acid. Methods for preparationof such formulations will be apparent to those skilled in the art. Thematerials also can be obtained commercially from Alza Corporation andNova Pharmaceuticals, Inc. Liposomal suspensions can also be used aspharmaceutically acceptable carriers. These can be prepared according tomethods known to those skilled in the art, for example, as described inU.S. Pat. No. 4,522,811. Microsomes and microparticles also can be used.

[0426] Oral or parenteral compositions can be formulated in dosage unitform for ease of administration and uniformity of dosage. Dosage unitform refers to physically discrete units suited as unitary dosages forthe subject to be treated; each unit containing a predetermined quantityof active compound calculated to produce the desired therapeutic effectin association with the required pharmaceutical carrier. Thespecification for the dosage unit forms of the invention are dictated byand directly dependent on the unique characteristics of the activecompound and the particular therapeutic effect to be achieved, and thelimitations inherent in the art of compounding such an active compoundfor the treatment of individuals.

[0427] As noted above, drugs identified or designed according to theinvention can be formulated into pharmaceutical compositions byadmixture with pharmaceutically acceptable nontoxic excipients andcarriers. Such compositions can be prepared for parenteraladministration, particularly in the form of liquid solutions orsuspensions; for oral administration, particularly in the form oftablets or capsules; or intranasally, particularly in the form ofpowders, nasal drops or aerosols. Where adhesion to a tissue surface isdesired the composition can include the drug dispersed in afibrinogen-thrombin composition or other bioadhesive. The drug then canbe painted, sprayed or otherwise applied to the desired tissue surface.Alternatively, the drugs can be formulated for parenteral or oraladministration to humans or other mammals, for example, intherapeutically effective amounts, e.g., amounts which provideappropriate concentrations of the drug to target tissue for a timesufficient to induce the desired effect.

[0428] Where the active compound is to be used as part of a transplantprocedure, it can be provided to the living tissue or organ to betransplanted prior to removal of tissue or organ from the donor. Thedrug can be provided to the donor host. Alternatively or, in addition,once removed from the donor, the organ or living tissue can be placed ina preservation solution containing the active compound. In all cases,the active compound can be administered directly to the desired tissue,as by injection to the tissue, or it can be provided systemically,either by oral or parenteral administration, using any of the methodsand formulations described herein and/or known in the art.

[0429] Where the drug comprises part of a tissue or organ preservationsolution, any commercially available preservation solution can be usedto advantage. For example, useful solutions known in the art includeCollins solution, Wisconsin solution, Belzer solution, Eurocollinssolution and lactated Ringer's solution.

[0430] The effective concentration of the compounds to be delivered in atherapeutic composition will vary depending upon a number of factors,including the final desired dosage of the compound to be administeredand the route of administration. The preferred dosage to be administeredalso is likely to depend on such variables as the type and extent ofdisease or indication to be treated, the overall health status of theparticular patient, the relative biological efficacy of the compounddelivered, the formulation of the drug, the presence and types ofexcipients in the formulation, and the route of administration. Ingeneral terms, the drugs of this invention can be provided to anindividual using typical dose units deduced from the earlier-describedmammalian studies using non-human primates and rodents.

[0431] When the active compounds are nucleic acid molecules, the nucleicacid may be inserted into vectors and used as gene therapy vectors. Genetherapy vectors can be delivered to a subject by, for example,intravenous injection, local administration (see U.S. Pat. No.5,328,470) or by stereotactic injection (see e.g., Chen et al. (1994)Proc. Natl. Acad. Sci. USA 91:3054-3057). The pharmaceutical preparationof the gene therapy vector can include the gene therapy vector in anacceptable diluent, or can comprise a slow release matrix in which thegene delivery vehicle is imbedded. Alternatively, where the completegene delivery vector can be produced intact from recombinant cells, e.g.retroviral vectors, the pharmaceutical preparation can include one ormore cells which produce the gene delivery system.

[0432] When an active compound of the invention is intended foradministration to a plant host, the invention may be applied directly tothe plant environment, for example, to the surface of leaves, buds,roots or floral parts. Alternatively, the present invention can be usedas a seed coating. The determination of an effective amount of thepresent invention as required for a particular plant is within the skillof the art and will depend on such factors as the plant species, methodof planting, and soil type. It is contemplated that compositionscontaining drugs of the invention can be prepared by formulating suchdrugs with adjuvants, diluents, carriers, etc., to provide compositionsin the form of filings/divided particulate solids, granules, pellets,wetable powders, dust, aqueous suspensions or dispersions, andemulsions. It is further contemplated to use such drugs in capsulatedform, for example, the drugs can be encapsulated within polymer,gelatin, lipids or other formulation aids such as emulsifiers,surfactants wetting agents, antifoam agents and anti-freeze agents, maybe incorporated into such compositions especially if such compositionswill be stored for any period of time prior to use. Application ofcompositions containing drugs of the invention as the active agent canbe carried out by conventional techniques. When an active compound isintended for administration to an insect host, standard methods such as,but not limited to, aerial dispersal are contemplated.

[0433] Active compound identified or designed by a method of theinvention also include precursors of the active compounds. The termprecursors refers to a pharmacologically inactive (or partiallyinactive) derivative of a parent molecule that requiresbiotransformation, either spontaneous or enzymatic, within the organismto release the active compounds. Precursors are variations orderivatives of the compounds of the invention which have groupscleavable under metabolic conditions. Precursors become the activecompounds of the invention which are pharmaceutically active in vivo,when they undergo solvolysis under physiological conditions or undergoenzymatic degradation. Precursor forms often offer advantages ofsolubility, tissue compatibility, or delayed release in the mammalianorganism (see Bundgard, Design of Prodrugs, pp. 7-9, 21-24, Elsevier,Amsterdam (1985); and Silverman, The Organic Chemistry of Drug Designand Drug Action, pp. 352-401, Academic Press, San Diego, Calif. (1992).

[0434] Active compound as identified or designed by the methodsdescribed herein can be administered to individuals to treat disorders(prophylactically or therapeutically). In conjunction with suchtreatment, pharmacogenomics (i.e., the study of the relationship betweenan individual's genotype and that individual's response to a foreigncompound or drug) may be considered. Differences in metabolism oftherapeutics can lead to severe toxicity or therapeutic failure byaltering the relation between dose and blood concentration of thepharmacologically active drug. Thus, a physician or clinician mayconsider applying knowledge obtained in relevant pharmacogenomicsstudies in determining whether to administer a drug as well as tailoringthe dosage and/or therapeutic regimen of treatment with the drug.

[0435] With regard to mammals, it is contemplated that the effectivedose of a protein synthesis inducer or inhibitor will be in the range ofabout 0.01 to about 50 mg/kg, preferably about 0.1 to about 10 mg/kg ofbody weight, administered in single or multiple doses. Typically, theinducer or inhibitor may be administered to a human recipient in need oftreatment at a daily dose range of about 1 to about 2000 mg per patient.

[0436] In light of the foregoing general discussion, the specificexamples presented below are illustrative only and are not intended tolimit the scope of the invention. Other generic and specificconfigurations will be apparent to those persons skilled in the art.

III. EXAMPLES A. Example 1

[0437] Preparation of 50S Ribosomal Subunit Crystals

[0438]H. marismortui (ATCC 43049) was grown as described previously (Banet al. (1998) supra) on a slightly modified version of ATCC culturemedium 1230, which was supplemented with 4.3 g of yeast extract, 5.1 gof Tris, and 3.4 g of glucose per liter. Bacteria were grown at 37° C.to an OD_(550nm) between 1.0 and 2.2. They were harvested bycentrifugation, and stored at −80° C. Cells were ruptured using a Frenchpress. Ribosomes were prepared from lysates by centrifugation, andsubunits were isolated on sucrose gradients (Shevack et al. (1985) FEBSLett. 184: 68-71).

[0439] 1. Reverse Extraction

[0440] (1) Take 1 mg of subunits from a concentrated 50S ribosomalsubunit stock (30 mg/ml in 1.2 M KCl, 0.5 M NH4Cl, 20 mM MgCl₂, Tris 10mM, CdCl₂ 1 mM, Tris 5 mM, pH 7.5) and mix with ½ vol. of 30% PEG6000(300 g PEG, 700 ml H₂O to make 1 liter of 30% PEG; filter through 0.2 μmfilter). Leave on ice for 1 to 2 hr.

[0441] (2) Spin down precipitate for about 30 seconds using a desktopcentrifuge.

[0442] (3) Remove supernatant and add 100 μl of RE-buffer: 7% PEG6000,1.2 M KCl, 0.5 M NH₄Cl, 100 mM KAc, 30 mM MgCl₂, 10 mM Tris, 10 mM MES(pH 7.5), and 1 mM CdCl₂

[0443] (4) Resuspend pellet at room temperature by mixing with a P200pipette set at 50 μl. Resuspended material should appear a littlecloudy.

[0444] (5) Wrap the eppendorf tube in aluminum foil and leave forequilibration at room temperature for 30-60 min. The solution will besaturated with 50S.

[0445] (6) Spin down for 2 minutes in desk-top centrifuge at roomtemperature, transfer supernatant to new eppendorf tube. A little pelletshould be found in the tube used for centrifugation. Keep thesupernatant at room temperature.

[0446] (7) Put 8-10 μl of supernatant in the sample well of a sittingdrop tray (Charles-Supper). Streak seed one hour later from a seedstock. Seed stock is prepared by putting previously grown crystals instabilizing solution buffer A (see below), and then vortexing themviolently. To streak seed, a human hair cleaned with water and ethanoland then dried is passed through the vortexed solution and then touchedon the new crystallization drop. Drops should look cloudy. Thereservoirs in the sitting drop trays contain 1000 μl of a solutioncontaining 8% PEG6000, 1.2 M KCl, 0.5 M NH₄Cl, 100 mM KAc, 6.5 mM HAc(yields pH 5.8), 30 mM MgCl₂, and 1 mM CdCl₂.

[0447] (8) Check after one day if seeding is succeeded. If yes, letcrystals grow for three weeks.

[0448] 2. Stabilization Protocol

[0449] When crystals have finished growing (after approximately 3weeks), each sitting drop chamber is opened by making just a single cut(slit) going from the middle and to the edge of the well. Through thisnarrow slit, 10 μl of buffer A (1.2 M KCl, 0.5 M NH4Cl, 30 mM MgCl₂, 10%PEG6000, 1 mM CdCl₂, 100 mM KAc, 10 mM Tris (titrated to final pH 6.1),30 mM MES) at room temperature is added to each drop and 45 μl of BufferC (0.667 M MES, 0.333 M Tris) to each reservoir.

[0450] Trays are put in a plastic box with a lid, and put in a 16° C.incubator for approximately one day, and then moved to 12° C. foranother day. The plastic box is then put in a polystyrene container witha lid, and put in the cold room for yet another day. Crystals can bekept like this for a long time, but need to undergo a further changingof buffer prior to any use.

[0451] Make the following transition series using buffer A and buffer B(1.7 M NaCl, 0.5 M NH₄Cl, 30 mM MgCl₂, 1 mM CdCl₂, 12% PEG6000, 20% EG,100 MM KAC (titrated to final pH 5.8 with HAC) to give final ratios ofbuffer B to buffer A of: {fraction (1/16)}, ⅛, ¼, ½, ¾. All solutionsshould be at cold room temperature. All manipulations of the drops willtake place through the narrow slit.

[0452] (1) Add 40 μl “{fraction (1/16)}” to the drop, leave for 15minutes.

[0453] (2) Add 40 μl “⅛” to the drop, leave for 30-60 minutes.

[0454] (3) Take out 40 μl from the drop (and discard it in thereservoir), add 40 μl “¼”, leave for 30-60 minutes.

[0455] (4) Take out 40 μl from the drop (and discard it in thereservoir), add 40 μl “½”, leave for 15 minutes.

[0456] (5) Take out 40 μl from the drop (and discard it in thereservoir), add 40 μl “¾”, leave for 15 minutes.

[0457] (6) Take out 40 μl from the drop (and discard it in thereservoir), add 40 μl buffer B, leave for 15 minutes.

[0458] (7) Take out 60-80 μl from the drop (and discard it in thereservoir), add 60-80 μl Buffer B, replace reservoirs with 500 μl bufferB.

B. Example 2

[0459] Determination of the Crystal Structure of the 50S RibosomalSubunit, With the Initial Refinement

[0460] All data, except the two native data sets, were collected at theNational Synchrotron Light Source (Brookhaven) from crystals frozen at100 K, using beamlines X12b and X25 and recorded using a 345 mm MARimaging plate. For each heavy atom derivative, anomalous diffractiondata were collected at the wavelength corresponding to the peakanomalous scattering. The beam size was 100×100 μm for most datacollections at X25 and 200×200 μm at beamline X12b. The crystals werealigned along the long axis of the unit cell (570 Å) so that 1.0°oscillations could be used to collect reflections out to a maximum of2.7 Å resolution at the edge of the MAR detector. At beamline X12b thecrystal to detector distances varied between 450.0 mm and 550.0 mmdepending on wavelength, crystal quality and beam divergence, and it waschosen so that maximum resolution data could be collected while avoidingoverlapping of spots. At beamline X25 the detector was positioned on arigid platform at 480 mm which allowed data collection to 3.2 Å foriridium and osmium derivatives with the wavelength set at the anomalousedge. Native data to 2.4 Å resolution were collected at the structuralbiology beamline ID19 of the Advanced Photon Source (Argonne) using aCCD detector (etc.). Data sets were processed by using DENZO andSCALEPACK (Otwinowski, (1993) Data Collection and Processing).

[0461] Heavy atom based phasing was extended to 3.2 Å resolution bycombining MIR phases calculated for two different isomorphous groups ofdata (MIR1 and MIR2, Table 1) with single derivative anomalousdispersion (SAD) phases. The best two derivatives were osmium pentamineand iridium hexamine, each of which contained a large number of bindingsites (Table 1). Several other derivatives with smaller number of sitesfurther improved map quality. All phasing was done by maximum likelihoodmethod implemented in CNS (Brünger et al. (1998) supra) with theexception of the Ta₆Br₁₂ derivative, which was refined in SHARP (de LaFortelle, (1997) Meth. Enzymol. 276: 472-494) represented as sphericallyaveraged electron density (Table 1). Phases were improved and extendedfrom 3.3 Å to 2.4 Å by solvent flipping (Abrahams et al. (1996) supra)and models were built.

C. Example 3

[0462] Preparation of Crystals of 50S Ribosomal Subunit/PuromycinComplex and Collection of X-ray Diffraction Data

[0463] Crystals of 50S ribosomal subunits were grown and stabilized asdescribed earlier. CCdA-p-puromycin (see FIG. 9A) was a generous giftfrom Michael Yarus (Welch, et al. (1995) supra). Oligonucleotides fromamino-N-acylated minihelices (see FIG. 9B) were synthesized byDharmacon. Following deprotection, the oligonucleotides were heatedbriefly to 100° C. and snap-cooled on ice to reanneal. Ribosomal 50Ssubunit crystals were stabilized and then soaked for 24 hours instabilization buffer plus 100 μM CCdA-p-puromycin or amino-N-acylatedmini-helices prior to cryovitrification in liquid propane and X-raydiffraction data collection. Phases were calculated by densitymodification (CNS) beginning with the best experimental phases using2F_(o)(analogue)-F_(o)(native) for amplitudes, from 60.0 to 3.2 Å.(Native amplitudes were from the most isomorphous native 1 data set,except for those amplitudes which were present only in the more completenative 2 data set. Calculated 2F_(o)-F_(o) amplitudes which were lessthan twice the corresponding calculated & were replaced byF_(o)(analogue)). Maps were then calculated using phases from densitymodified and 2F_(o)(analogue)-F_(o)(native) orF_(o)(analogue)-F_(o)(native) amplitudes.

D. Example 4

[0464] Antibiotic Binding Sites Located in the Polypeptide Exit TunnelNear the Peptidyl Transferase Center

[0465] Crystalline complexes of the H. marismortui large subunitcomplexed with three antibiotics have been established at about 3.0 Åresolution. The electron density maps at this resolution have allowed usto position approximately on the ribosome the antibiotics tylosin,carbomycin and anisomycin. We observed that these antibiotics all bindto the ribosome in the region that lies between the peptidyl transferasecenter as defined by the Yarus inhibitor, CCdA-p-puromycin, and the tipsof the proteins L22 and L4 at the point that they form a small orificein the polypeptide exit tunnel. The general location of this majorantibiotic binding site is shown in FIG. 19. Tylosin and carbomycinappear to function by blocking the exit of newly synthesizedpolypeptides. Anisomycin blocks the A site.

[0466] It is contemplated that the antibiotic erythromycin will bind inalmost the same location as tylosin because of the similarity of the twomolecules and because erythromycin resistance mutations are known inboth the tip of protein L4 and in portions of the RNA near the tylosinbinding site.

[0467] The vast majority of the interactions between those antibioticsand the ribosome are through rRNA that forms the A site, and the surfaceof the tunnel between the peptidyl transferase center and protein L22.Since these antibiotics do not bind identically, there will be manyadditional ways that small molecule compounds can be designed to bind inthis region using the tools and methodologies of the present invention.For example, by connecting together components of each of the differentantibiotics which bind to non-overlapping sites it will be possible tocreate new antibiotics (see, Example 6). In addition, based on newprinciples of small molecule RNA interaction shown by these antibioticcomplexes we will be able to design entirely novel small molecules thatwill bind to sites on the ribosome as well as other potential RNAtargets.

E. Example 5

[0468] Design and Testing of Hybrid Antibiotics

[0469] Many antibiotics that target ribosomes, more particularly largeribosomal subunits, and disrupt protein synthesis are complex moleculesthat are effectively concatenations of simpler substructures, at leastone of which interacts with a discrete part of the ribosome. When thecompound in question includes several interactive substructures, itsbinding site is effectively the sum of the subsites that contact andengage each such substructure. It has been found that many antibioticsthat target the large ribosomal subunit bind the ribosomal subunit atsites that are close to one another. Thus the possibility exists ofsynthesizing new antibiotics in which one ribosome-binding moiety of afirst known antibiotic is linked chemically to a ribosome-binding moietyof a second known antibiotic that interacts with an adjacent subsite.The new compound that results is thus a chimera of the two antibioticsfrom which it derives.

[0470] Chimeric antibiotics can be designed using the information aboutthe structures of antibiotic/ribosome complexes discussed hereinabove.These structures permit the identification of antibiotic bindingsubsites in the ribosome, and the specification of the chemical entitiesthat interact with them. Equipped with such knowledge, those skilled inthe art of organic synthesis can synthesize compounds that link thesubstructures of interest together in ways that should enable them tointeract with their respective subsites at the same time. Any compounddevised this way that functions in the manner intended is likely toinhibit cell growth and if it does, protein synthesis in vivo. At thevery least, it should block protein synthesis in in vitro assay systems.Further information about the ribosomal interactions of such a compoundcan be obtained by determining the structure of the complex it formswith the ribosome using the methods described in Section D, hereinabove.

[0471] For example, as a result of the work described herein, it hasbeen discovered that the disaccharide moiety of carbomycin binds thelarge ribosomal subunit at a site in close proximity to the binding sitefor a portion of the anisomycin. Using this information and the softwarepackages described hereinabove, the skilled artisan can design a hybridantibiotic comprising the relevant ribosome binding portions ofcarbomycin and anisomycin linked by a suitable chemical linker. Thishybrid molecule, once designed, can be synthesized and purified usingconventional synthetic organic chemistries and conventional purificationschemes. Once synthesized and purified, the hybrid molecule can bescreened for bioactivity. These screens can include, for example,growing micro-organisms on or in media either supplemented or lackingthe hybrid molecule. Any reduction in the number of micro-organisms orthe size of colonies in the presence of the hybrid molecule would beindicative of bioactivity. Furthermore, the hybrid molecule could betested in a cell free translation system in the presence of one or morelabeled amino acids. Any reduction in the level of labeled amino acidsincorporated into proteins in cell free systems that include the hybridmolecule relative to cell free systems locking the hybrid molecule wouldbe indicative that the hybrid molecule acts as a functional proteinsynthesis inhibitor. It is contemplated that the hybrid molecule couldthen be iteratively refined as discussed hereinabove to enhance itsbioactive peptides and bioavailability.

INCORPORATION BY REFERENCE

[0472] The disclosure of each of the patent documents, scientificarticles, atomic-co-ordinates (including, without limitation, those setsdeposited at the Research Collaboratory for Structural BioinformaticsProtein Data Bank (PDB) with the accession numbers PDB ID: 1FFK; PDB ID:1FF2; PDB ID: 1FG0; and PDB ID: 1JJ2, and/or contained on Disk No. 1, 2,or 3) referred to herein is incorporated by reference herein.

[0473] All materials submitted herewith on Disk Nos. 1, 2 and 3 areincorporated by reference herein. Disk Nos. 1, 2 and 3 are identified ascontaining the following files: File Name: Size: Date Created Disk No. 1of 3: 1) 1ffk.doc 7,046 KB 7/25/01 2) 1ffk.ent 5,484 KB 7/25/01 3)1ffz.doc 1,219 KB 7/25/01 4) 1ffz.ent 937 KB 7/25/01 5) 1fg0.doc 1,225KB 7/25/01 6) 1fg0.ent 942 KB 7/25/01 7) <dir> 7/25/01 8) <dir> 7/25/01Disk No. 2 of 2: 1) 1jj2.rtf 12,742 KB 7/25/01 2) 1jj2.txt 8,372 KB7/26/01 3) <dir> 7/26/01 4) <dir> 7/26/01 Disk No. 3 of 3: 1)anisomycin.pdb 7,593 KB 7/18/01 2) blasticidin.pdb 7,594 KB 7/18/01 3)carbomycin.pdb 7,592 KB 7/18/01 4) sparsomycin.pdb 7,541 KB 7/18/01 5)spiramycin.pdb 7,592 KB 7/18/01 6) tylosin.pdb 7,592 KB 7/18/01 7)virginiamycin.pdb 7,591 KB 7/18/01 8) <dir> 7/18/01 9) <dir> 7/18/01

EQUIVALENTS

[0474] The invention may be embodied in other specific forms withoutdeparting form the spirit or essential characteristics thereof. Theforegoing embodiments are therefore to be considered in all respectsillustrative rather than limiting on the invention described herein.Scope of the invention is thus indicated by the appended claims ratherthan by the foregoing description, and all changes that come within themeaning and range of equivalency of the claims are intended to beembraced therein.

1 1 1 33 RNA Artificial sequence oligonucleotide sequence that shouldform 12 base pairs 1 ccggcgggcu gguucaaacc ggcccgccgg acc 33

What is claimed is:
 1. A crystal of a ribosome or a ribosomal subunitwherein the crystal has an average thickness greater than about 15 μm.2. The crystal of claim 1 wherein the average thickness is selected fromthe group consisting of from about 16 μm to about 65 μm, from about 66μm to about 105 μm, from about 104 μm to about 155 μm, and from about156 μm to about 205 μm.
 3. The crystal of claim 1 wherein the averagethickness is from about 100 μm to about 200 μm.
 4. An untwinned crystalof a ribosome or ribosomal subunit.
 5. The crystal of claim 1 or 4wherein the ribosomal subunit is a large ribosomal subunit.
 6. Thecrystal of claim 1 or 4 wherein the ribosomal subunit is a smallribosomal subunit.
 7. The crystal of claim 1 or 4 wherein the ribosomalsubunit is a 50S ribosomal subunit.
 8. The crystal of claim 1 or 4wherein the ribosome or ribosomal subunit is obtained from a prokaryoteor from an eukaryote.
 9. The crystal of claim 1 or 4 wherein theribosome or ribosomal subunit is obtained from an archaebacteria. 10.The crystal of claim 1 or 4 wherein the ribosome or ribosomal subunit isobtained from Haloarcula marismortui.
 11. The crystal of claim 1 or 4wherein the ribosomal subunit is a 60S ribosomal subunit.
 12. Thecrystal of claim 1 or 4 wherein the ribosome or ribosomal subunit isobtained from a mammal.
 13. The crystal of claim 1 or 4 wherein thecrystal effectively diffracts X-rays for determination of atomicco-ordinates to a resolution of at least about 3.0 Å.
 14. The crystal ofclaim 1 or 4 further comprising a ligand.
 15. The crystal of claim 14wherein the ligand is bound to the ribosome or the ribosomal subunit.16. The crystal of claim 15 wherein the ligand is an antibiotic.
 17. Thecrystal of claim 16 wherein the antibiotic is a macrolide antibiotic.18. A crystal of a ribosome or a ribosomal subunit wherein the crystaleffectively diffracts X-rays for determination of atomic co-ordinates toa resolution of at least about 3.0 Å.
 19. A crystal of a ribosome or aribosomal subunit wherein the crystal effectively diffracts X-rays fordetermination of atomic co-ordinates to a resolution of about 2.4 Å. 20.A crystal of a ribosome or a ribosomal subunit wherein the crystal issufficient to determine the atomic co-ordinates of the ribosome orribosomal subunit.
 21. A crystal of a 50S ribosomal subunit comprisingan atomic structure characterized by the atomic co-ordinates depositedat the Protein Data Bank under accession number PDB ID: 1FFK or 1JJ2.22. Phases computed from the co-ordinates of claim
 21. 23. A method ofobtaining an electron density map of a selected ribosomal subunit,wherein the selected ribosomal subunit is different from the ribosomalsubunit used to obtain the computed phases of claim 22, said methodcomprising: (a) producing a crystal of a selected ribosomal subunit,wherein the crystal is isomorphous; (b) obtaining diffraction amplitudesof the crystal produced in step (a); (c) combining the computed phasesof claim 22 with the diffraction amplitudes obtained in step (b) toproduce a combined data set; and (d) obtaining an electron density mapof the selected ribosomal subunit based on the combined data setobtained in step (c).
 24. A method of obtaining an electron density mapof a selected ribosomal subunit, wherein the selected ribosomal subunitis closely related to the ribosomal subunit used to obtain the computedphases of claim 22, said method comprising: (a) producing a crystal of aselected ribosomal subunit, wherein the crystal crystallizes in adifferent unit cell with different symmetry than the crystal which wasused to compute the phases of claim 22; (b) obtaining X-ray diffractiondata for the crystal produced in step (a); (c) obtaining phases of theselected ribosomal subunit by using the data obtained in step (b) andthe computed phases of claim 22 in a molecular replacement technique;and (d) obtaining an electron density map of the selected ribosomalsubunit from the phases obtained in step (c).
 25. A method of obtaininga model of a selected ribosomal subunit, wherein the selected ribosomalsubunit diverges from but is still homologous to the ribosomal subunitused to obtain the computed phases of claim 22, said method comprising:(a) producing a crystal of a selected ribosomal subunit; (b) obtainingatomic co-ordinates for the crystal produced in step (a); (c) obtaininga model for the selected ribosomal subunit by homology modeling usingthe atomic co-ordinates obtained in step (b) and the computed phases ofclaim
 22. 26. A method of growing a crystal of a ribosome or a ribosomalsubunit comprising: (a) isolating a ribosome or a ribosomal subunit; (b)precipitating the ribosome or ribosomal subunit; (c) back-extracting theprecipitated ribosome or ribosomal subunit to obtain a solution; (d)seeding the back-extracted solution; (e) growing a crystal of theribosome or ribosomal subunit from the seeded solution by vapordiffusion at room temperature; and (f) harvesting the crystal.
 27. Themethod of claim 26 further comprising: (g) stabilizing the crystal bygradual transfer into a solution containing a high salt concentration;and (h) maintaining the crystal under high salt concentration.
 28. Themethod of claim 27 wherein the high salt concentration is from about 1.2M salt to about 1.7 M salt.
 29. The method of claim 27 furthercomprising: (i) flash freezing the crystal.
 30. A crystal produced bythe method of claim 26, 27, 28 or
 29. 31. A method of obtaining X-raydiffraction data for a crystal of a ribosome or a ribosomal subunitcomprising: (a) obtaining a crystal of a ribosome or a ribosomalsubunit, wherein the crystal has one or more of the followingcharacteristics: (1) an average thickness of greater than 15 μm; (2)untwinned; and (b) using X-ray crystallography to obtain X-raydiffraction data for the crystal of the ribosome or ribosomal subunit.32. A method of obtaining an electron density map of a ribosome or aribosomal subunit comprising using the X-ray diffraction data obtainedby the method of claim 31 to obtain an electron density map of theribosome or ribosomal subunit.
 33. A method of obtaining X-raydiffraction data for a complex of a ribosome and a ligand or a complexof a ribosomal subunit and a ligand comprising: (a) obtaining a crystalof a ribosome or a ribosomal subunit, wherein the crystal has one ormore of the following characteristics: (1) an average thickness ofgreater than 15 μm; (2) untwinned; (b) diffusing a ligand through thecrystal so that the ligand binds the ribosome or ribosomal subunit toform a complex; and (c) using X-ray crystallography to obtain X-raydiffraction data for the complex.
 34. A method of obtaining X-raydiffraction data for a complex of a ribosome and a ligand or for aribosomal subunit and a ligand comprising: (a) obtaining a co-crystalfor a complex of a ribosome and a ligand or for a complex of a ribosomalsubunit and a ligand, wherein the co-crystal has one or more of thefollowing characteristics: (1) an average thickness of greater than 15μm; (2) untwinned; and (b) using X-ray crystallography to obtain X-raydiffraction data for the complex.
 35. A method of obtaining an electrondensity map for a complex of a ribosome and a ligand or for a complex ofa ribosomal subunit and a ligand comprising using the X-ray diffractiondata obtained by the method of claim 33 or 34 to obtain an electrondensity map of the complex of the ribosome and the ligand or for thecomplex of the ribosomal subunit and the ligand.
 36. The method of claim33 or 34 wherein the ligand is an antibiotic.
 37. A method of locatingthe attachment of a ligand to a ribosome or the attachment of a ligandto a ribosomal subunit comprising: (a) obtaining X-ray diffraction datafor a ribosome or for a ribosomal subunit according to claim 31; (b)obtaining X-ray diffraction data for a complex of a ribosome and aligand or for a complex of a ribosomal subunit and a ligand according tothe method of claim 33 or 34; (c) subtracting the X-ray diffraction dataobtained in step (a) from the X-ray diffraction data obtained in step(b) to obtain the difference in the X-ray diffraction data; (d)obtaining phases that correspond to X-ray diffraction data obtained instep (a) using one or more of the techniques selected from the groupconsisting of MIR, MIRAS and SAD; (e) utilizing the phases obtained instep (d) and the difference in the X-ray diffraction data obtained instep (c) to compute a difference Fourier image of the ligand; and (f)locating the attachment of the ligand to a ribosome or the attachment ofthe ligand to a ribosomal subunit based on the computations obtained instep (e).
 38. A method of obtaining a map of a ligand attached to aribosome or of a ligand attached to a ribosomal subunit comprising: (a)obtaining X-ray diffraction data for a ribosome or for a ribosomalsubunit according to claim 31; (b) obtaining X-ray diffraction data fora complex of a ribosome and a ligand or a complex of a ribosomal subunitand a ligand according to the method of claim 33 or 34; (c) obtainingphases that correspond to X-ray diffraction data obtained in step (a)using one or more of the techniques selected from the group consistingof MIR, MIRAS and SAD; and (d) utilizing the phases obtained in step (c)and the X-ray diffraction data obtained in step (b) to compute a map ofthe ligand and the ribosome or of the ligand and the ribosomal subunit.39. The method of claim 37, wherein the ligand is an antibiotic.
 40. Amethod of obtaining a modified agent comprising: (a) obtaining a crystalof a ribosome or of a ribosomal subunit with or without a bound agent;(b) obtaining the atomic co-ordinates of at least a portion of theribosome or ribosomal subunit with or without the bound agent; (c) usingthe atomic co-ordinates and one or more molecular modeling techniques todetermine how to modify the interaction of the agent with a ribosome orribosomal subunit; and (d) modifying the agent based on thedeterminations obtained in step (c) to produce a modified agent.
 41. Themethod of claim 40, wherein the one or more molecular modelingtechniques are selected from the group consisting of graphic molecularmodeling and computational chemistry.
 42. The method of claim 40 furthercomprising contacting the modified agent with a ribosome or ribosomalsubunit and detecting the interaction of the modified agent to theribosome or ribosomal subunit.
 43. A modified agent produced by themethod of claim 40 wherein the modified agent binds differently to aribosome or ribosomal subunit than does the agent from which themodified agent was derived.
 44. The modified agent of claim 43, whereinthe modified agent is a therapeutic agent.
 45. The method of claim 40,wherein the atomic co-ordinates of the ribosome or ribosomal subunitcrystal are deposited at the Protein Data Bank under accession numberPDB ID: 1FFK, 1FFZ, 1FG0 or 1JJ2.
 46. A modified agent produced by themethod of claim 40, wherein the modified agent binds differently to aribosome or ribosomal subunit than does the agent from which themodified agent was derived.
 47. A computer system comprising: (a) amemory having stored therein data indicative of atomic co-ordinatesderived from an electron density map having a resolution of at leastabout 4.5 Å and defining a ribofunctional locus of a large subunit of aribosome; and (b) a processor in electrical communication with thememory, the processor comprising a program for generating athree-dimensional model representative of the ribofunctional locus. 48.The computer system of claim 47, further comprising a device forproviding a visual representation of the model.
 49. The computer systemof claim 47, wherein the atomic co-ordinates comprise at least a portionof the atomic co-ordinates deposited at the Protein Data Bank underaccession number PDB ID: 1FFK, 1FFZ, 1FG0, or 1JJ2.
 50. The computersystem of claim 47, wherein the atomic co-ordinates further define atleast a portion of a protein synthesis inhibitor complexed with aribofunctional locus.
 51. The computer system of claim 50, wherein theprotein synthesis inhibitor is an antibiotic.
 52. The computer system ofclaim 51, wherein the atomic co-ordinates comprise at least a portion ofthe atomic co-ordinates recorded on Disk No. 3 of 3 under file numberanisomycin.pdb, blasticidin.pdb, carbomycin.pdb, sparsomycin.pdb,spiramycin.pdb, tylosin.pdb, or virginiamycin.pdb.
 53. The computersystem of claim 47, wherein the ribofunctional locus comprises at leasta portion of an active site in the ribosomal subunit.
 54. The computersystem of claim 53, wherein the active site comprises at least a portionof a peptidyl transferase site.
 55. The computer system of claim 54,wherein the peptidyl transferase site is defined by a plurality ofresidues set forth in Table
 5. 56. The computer system of claim 47,wherein the ribofunctional locus comprises at least a portion of anA-site.
 57. The computer system of claim 56, wherein the A-site isdefined by a plurality of residues set forth in Table
 6. 58. Thecomputer system of claim 47 or 56, wherein the ribofunctional locuscomprises at least a portion of a P-site.
 59. The computer system ofclaim 58, wherein the P-site is defined by a plurality of residues setforth in Table
 7. 60. The computer system of claim 47 or 56, wherein theribofunctional locus comprises at least a portion of a polypeptide exittunnel.
 61. The computer system of claim 60, wherein the exit tunnel isdefined by a plurality of residues set forth in Table 8, Table 9 orTable
 10. 62. The computer system of claim 58, wherein theribofunctional locus comprises at least a portion of a polypeptide exittunnel.
 63. The computer system of claim 62, where the exit tunnel isdefined by a plurality of residues set forth in Table 8, Table 9 orTable
 10. 64. The computer system of claim 47, wherein theribofunctional locus is defined by a plurality of residues set forth inTable 11, Table 12, Table 13, Table 14, Table 15, Table 16 or Table 17.65. The computer system of claim 47, wherein the atomic co-ordinates areproduced by molecular modeling.
 66. The computer system of claim 47 or65, wherein the atomic co-ordinates are produced by homology modelingusing at least a portion of the atomic co-ordinates deposited at theProtein Data Bank under accession number PDB ID: 1FFK, 1FFZ, 1FG0, or1JJ2.
 67. The computer system of claim 47 or 65, wherein the atomicco-ordinates are produced by molecular replacement using at least aportion of the atomic co-ordinates deposited at the Protein Data Bankunder accession number PDB ID: 1FFK, 1FFZ, 1FG0, or 1JJ2.
 68. Thecomputer system of claim 47, wherein the ribofunctional locus is definedby atoms of a ribosomal RNA.
 69. The computer system of claim 47 or 68,wherein the ribofunctional locus is defined by atoms of a ribosomalprotein.
 70. The computer system of claim 47, wherein the atomicco-ordinates define a residue that is present in a ribosome of apathogen but absent from a ribosome of a host organism.
 71. The computersystem of claim 70, wherein the host organism is a mammal.
 72. Thecomputer system of claim 71, wherein the mammal is a human.
 73. Thecomputer system of claim 47, wherein the atomic co-ordinates defineresidues that are conserved among pathogens.
 74. The computer system ofclaim 47, further comprising a program for performing drug design.
 75. Amolecular model produced by the computer system of claim
 47. 76. Amethod of identifying a candidate molecule, the method comprising thesteps of: (a) providing a molecular model of a ribofunctional locus of alarge subunit of a ribosome, wherein the molecular model is based onatoms derived from an electron density map having a resolution of atleast about 4.5 Å; and (b) using the model to identify a candidatemolecule having a surface complementary to the ribofunctional locus. 77.The method of claim 76, wherein the candidate molecule binds theribofunctional locus of the large subunit of the ribosome.
 78. Themethod of claim 76, comprising the additional step of producing thecandidate molecule identified in step (b).
 79. The method of claim 76 or78, comprising the additional step of determining whether the candidatemolecule modulates ribosomal activity.
 80. The method of claim 79,comprising the additional step of identifying a modified molecule. 81.The method of claim 80, comprising the additional step of producing themodified molecule.
 82. The method of claim 81, comprising the additionalstep of determining whether the modified molecule modulates ribosomalactivity.
 83. The method of claim 82, comprising the additional step ofproducing the modified molecule.
 84. The method of claim 76, wherein thecandidate molecule is an antibiotic or an antibiotic analogue.
 85. Themethod of claim 80, wherein the modified molecule is an antibiotic or anantibiotic analogue.
 86. The method of claim 84, wherein the antibioticor antibiotic analogue is a macrolide.
 87. The method of claim 76,wherein the ribofunctional locus comprises at least a portion of anactive site.
 88. The method of claim 87, wherein the active sitecomprises at least a portion of a peptidyl transferase site.
 89. Themethod of claim 87, wherein the peptidyl transferase site is defined bya plurality of residues set forth in Table
 5. 90. The method of claim76, wherein the ribofunctional locus comprises at least a portion of anA-site.
 91. The method of claim 90, wherein the A-site is defined by aplurality of residues set forth in Table
 6. 92. The method of claim 76or 90, wherein the ribofunctional locus comprises a least a portion of aP-site.
 93. The method of claim 92, wherein the P-site is defined by aplurality of residues set forth in Table
 7. 94. The method of claim 76or 90, wherein the ribofunctional locus comprises at least a portion ofa polypeptide exit tunnel.
 95. The method of claim 94, wherein the exittunnel is defined by a plurality of residues set forth in Table 8, Table9 or Table
 10. 96. The method of claim 92, wherein the ribofunctionallocus comprises at least a portion of a polypeptide exit tunnel.
 97. Themethod of claim 96, wherein the exit tunnel is defined by a plurality ofresidues set forth in Table 8, Table 9 or Table
 10. 98. The method ofclaim 76, wherein the ribofunctional locus is defined by a plurality ofresidues set forth in Table 11, Table 12, Table 13, Table 14, Table 15,Table 16 or Table
 17. 99. The method of claim 76, wherein the molecularmodel is in an electronic form.
 100. The method of claim 76, wherein themolecular model is generated from atomic co-ordinates produced bymolecular modeling.
 101. The method of claim 76 or 100, wherein themolecular model is generated from atomic co-ordinates produced byhomology modeling using at least a portion of the atomic co-ordinatesdeposited at the Protein Data Bank under accession number PDB ID: 1FFK,1FFZ, 1FG0, or 1JJ2.
 102. The method of claim 76 or 100, wherein themolecular model is generated from atomic co-ordinates produced bymolecular replacement using at least a portion of the atomicco-ordinates deposited at the Protein Data Bank under accession numberPDB ID: 1FFK, 1FFZ, 1FG0, or 1JJ2.
 103. The method of claim 76, whereinthe molecular model comprises residues that are conserved amongprokaryotic organisms.
 104. The method of claim 76, wherein themolecular model comprises a residue that is present in a prokaryoticribosome but is absent from a eukaryotic ribosome.
 105. The method ofclaim 104, wherein the eukaryotic ribosome is a mammalian ribosome. 106.An protein synthesis inhibitor comprising: a first binding domain havinga surface that mimics or duplicates a surface of a known first moleculethat binds with a first contact site in a large ribosomal subunit; and asecond binding domain having a surface that mimics or duplicates asurface of a known second molecule that binds with a second contact sitein the ribosomal subunit, wherein the first domain is attached to thesecond domain so as to permit both the first domain and the seconddomain to bind with its respective contact site thereby to disruptprotein synthesis in a ribosomal subunit.
 107. The inhibitor of claim106, wherein the first molecule is a first antibiotic.
 108. Theinhibitor of claim 106, wherein the first antibiotic binds at least aportion of a ribofunctional locus.
 109. The inhibitor of claim 106 or107, wherein the second molecule is a second antibiotic.
 110. Theinhibitor of claim 109, wherein the second antibiotic binds at least aportion of a ribofunctional locus.
 111. An engineered, synthetic proteinsynthesis inhibitor, the inhibitor comprising: a binding domain having asurface that mimics or duplicates a surface of a known molecule whichbinds with a contact site in a ribosomal subunit; and an effector domainattached to the binding domain which, upon binding of the binding domainwith the contact site, occupies a space within or adjacent the ribosomalsubunit thereby to disrupt protein synthesis in the ribosomal subunit.112. The inhibitor of claim 111, wherein the surface of the bindingdomain mimics or duplicates a surface of a known antibiotic which bindswith the contact site.